Supplementary Figure 1.

A B M0(CTR) M1(IFNG) M2(IL-4) 40 n=6334 30 n=7159 n=7000 40 MTE=12.9 MTE=12.6 MTE=11.1 30 20 30 20 20 10 10

TSS enrichment TSS 10 TSS enrichment score 0 0 0 2.5 3 3.5 4 4.5 5 2.5 3 3.5 4 4.5 5 2.5 3 3.5 4 4.5 5 9 17 Log10(unique fragments/cell) UMAP2 C UMAP1

EGR footprint STAT6 footprint IRF footprint STAT1 footprint 0.8 1.0 0.6 M0(CTR) 0.75 M2(IL-4) 0.5 0.6 0.4 M1(IFNG) 0.50 0.4 0.0 0.2 0.25 0.2 -0.5 0.0 Norm. insertionsNorm. 0.00 0.0 -1.0 -0.2 -200 -100 0 100 200 -200 -100 0 100 200 -200 -100 0 100 200 -200 -100 0 100 200 Distance from motif center (bp)

Motif deviation score

D mRNA level F -1 1

0 10 5 STAT6 0 Tlr2 Arg1 Itgax 1 0 8 4 -1

-1 EGR2 0 -1 1 6 3 -2 IL-4

FC overCTR FC 4 2

2 Pseudotime -3 -1 Log 2 1 1 IRF1 0 IL-4 1 0

-4 0 0 -1

0 -1 STAT1 1 1 3 6 24 1 3 6 24 1 3 6 24 IFNG Time (h) Pseudotime E LOST Accessibility mRNA level 2 Ifitm2 0.5 Ifitm2 2 Cd14 0 Cd14 2 Hpgd 1 Hpgd 0.0 -1 0 1 1 1 -0.5 -2 -1 0 0 0

FC overCTR FC -1.0 -3 -2 FC overCTR FC FC overCTR FC 2 Genescore 2 2 -1 Genescore -1 Genescore -1 Log Log

-1.5 Log -4 -3

-2 IL-4 -2.0 -2 -5 -2 -4

1 3 6 24 1 3 6 24 1 3 6 24 Pseudotime Time (h) Pseudotime Time (h) Pseudotime Time (h)

EARLY Accessibility mRNA level 2 5 2 8 2 4 Egr2 Mgl2 4 1 1 6 1 3 3 0 0 4 0 2 2 FC overCTR FC overCTR FC overCTR FC Genescore 2 2 2 Genescore Genescore -1 -1 2 -1 1

Log 1 Log Log

-2 IL-4 0 -2 0 -2 0

1 3 6 24 1 3 6 24 1 3 6 24 Pseudotime Time (h) Pseudotime Time (h) Pseudotime Time (h)

LATE Accessibility mRNA level 2 Anxa2 1.5 2 Mmp12 3 2 Pparg 1.5

1 1.0 1 2 1 1.0

0 0.5 0 1 0 FC overCTR FC overCTR FC Genescore overCTR FC

2 2 2 0.5 Genescore -1 0.0 Genescore -1 0 -1 Log Log Log

-2 IL-4 -0.5 -2 -1 -2 0.0

1 3 6 24 1 3 6 24 1 3 6 24 Pseudotime Time (h) Pseudotime Time (h) Pseudotime Time (h) Supplementary Figure 2.

Induced Repressed Repressed (n=147) Induced (n=214) vs. vs. A B IL-4 IL-4 6 2.5 change Fold Arg1 Ifitm3 Retnla Lyz2 2.0 4 Mmp12 Ifi27l2a M1(IFNG) Ccl7 Maf 1.5 (1754) 2 M0(CTR) Gatm Cx3cr1 200 1.0 (1058) Mgl2 Itga6 value

- Ccl2 Hpgd Slc7a2 Pid1 Cd36 Spp1 Itgax Clec4d

adjustedp Rnf19b Cd14

10 Cd300ld Pf4

log Clic4 Selenop - 100 Anxa4 Mpeg1 Smap2 Cd93 Egr2 Marcksl1 Lgals3 Gas6 Sdc4 Igfbp4

UMAP2 M2(IL-4) Mrc1 Cxcl16 UMAP1 (1692) Prkcd Ptpn18 M2(IL-4) -1 0 1 2 Average log2 Fold Change

C integration score (scRNA-seq) Gene expression Repressed (n=212) Induced (n=492) Induced Repressed F10 Max Itgax vs. vs. IFNG IFNG Retnla 300 2.5

15 change Fold Egr2 Cxcl10 C5ar1 Gbp2 Hmox1 Mmp12 Min 2.0 Iigp1 10 Cd300lb Slc7a2 Cxcl9 Adam8 1.5 Mgl2 Ifi47 5 Cx3cr1 1.0

Ccl7 value Irgm1 Dhrs3 - * Hist1h2bf 200 Socs1 B4galt6 Ccl2 Samhd1 Plin2 Kif4 Irf8 Pid1 Fcgr4 F630028O10Rik * Cenpt adjustedp Gbp2 10 Gbp5 Fgd4 Igtp Lhfpl2 log

Gbp10 - Nampt Arl4c Ifit3 100 Slfn5 Apbb1ip Cxcl9 Ifi203 Pmp22 Ifi44 Irf1 Zmiz1 Mx1 Gbp7 Ifngr1 * Top2a Ccl12 Cxcr4 Ch25h Ch25h Rps29 Clec9a M1(IFNG) Cd274 Sh3bp5 Tnfsf15 -1 0 1 2 3 4 Ccl5 Average log2 Fold Change * Cdk1 C1 C2 C3 C4 C5 C6

M2(IL-4) M1(IFNG) M0(CTR)

D STAT6 EGR2 STAT6 motif EGR2 motif Dev. score Dev. score C1 C1 C2 C2 C3 C3 C4 C4 C5 C5 C6 C6 UMAP2 -1.0 2.0 UMAP2 -0.5 1.0 UMAP1 UMAP1 IRF1 STAT1 IRF1 motif Dev. score STAT1 motif Dev. score

C1 C1 C2 C2 C3 C3 C4 C4 C5 C5

C6 Motif deviation score C6

UMAP2 -2.0 3.0 -1.0 2.0 UMAP2 Min Max UMAP1 UMAP1 Supplementary Figure 3.

A C M2(IL-4) Egr2 Gene score Phase distribution of the clusters 1.0 1.75 G1 C6 G1 S C5 M0(CTR) S G2/M C4 G2/M Gene score C3 M1(IFNG) Log2(NC+1)

C2 UMAP2 Min Max UMAP1 C1 M2(IL-4) Egr2 1.00 0.75 0.50 0.25 0 Gene expression 0.5 2.5

G1

B S

G2/M

M2(IL-4) M1(IFNG) UMAP2 Gene expression UMAP1 #Counts G1 vs. S S vs. G1 G1 vs. S S vs. G1 Fold induction Fold Fold induction Fold 1.6 Hnrnpab 2.5 induction Fold Atp6v0d2 1.5 Slfn5 1810037l17Rik – 1.45 Min Max Gatm Lgals3 1.5 Mmp13 Cdkn1a – 1.38 Ranbp1 Anxa1 1.4 Ifit2 2.0 D Anp32b Ctss 1.4 Ifit1 Pa2g4 Ptpre Ifitm2 1.5 Uba2 S vs. G2/M Arhgap25 Samhd1 Kpnb1 M1(IFNG) Iigp1 Gatm – 1.45 Irf8 Gene score Ran G2/M vs. G1 Ddx17 1.6 3.2 Hsp90aa1 Fold induction Fold Cd274 1.8 Set Gatm Ptprc G2/M vs. G1 G1 Ccl2 Hnrnpf 1.6 Creb5 Ccl7 Psat1 Sp100 Ly6a – 1.33 Hnrnpab Bach1 Jpt1 – 1.30 1.4 S Ran S vs. G2/M Anxa3 Anp32b Irf8 Hspd1 – 1.36 Ranbp1 Gbp5 Gene score G2/M Cdca7 – 1.36 Mgl2 Rrbp1 G2/M vs. S Log2(NC+1) Kpnb1 G2/M vs. S Clic4 Ch25h Mt1 – 1.45 UMAP2 Themis2 Min Max Pcyox1l None Pdgfb Mt2 – 1.33 UMAP1 Irf8 Gene expression 9 21

G1 E S

G2/M

UMAP2 Gene expression UMAP1 #Counts

Min Max M0(CTR)

G1 73 S 12 G2/M 6.6 Supplementary Figure 4. B C A Top50 repressed Top50 induced Cell cycle sensitive gene expression (%) 400 Cxcl9 Ifi44 Gbp4 Gbp2 M1(IFNG) M2(IL-4) G1 0-140 0-320 0-1200 Cxcl10 Sensitive Sensitive 300 Irf8 Insensitive Insensitive M0(CTR) S Rps29 CTR 76% 74% G2/M 200 Cd300lb Cxcl9

Log_padj C5ar1 G1 -log_padj 100 Cx3cr1 Induced Repressed Induced Repressed Sensitive Sensitive S Sensitive Insensitive Insensitive Sensitive Insensitive Insensitive Plin2 IFNG M1(IFNG) G2/M 0 87% 68% 66% 82% -5 0 5 Avg.Avg. loglogFC2FC D E IFNG-induced IFNG-repressed IL-4-repressed Cell cycle sensitive Cell cycle sensitive Cell cycle sensitive 2 Ctsc Ifngr1 Sesn1 2 2 Z Irf8 Z Fgd4 Z Wwp1 - - - scores

1 scores scores Samhd1 Plin2 C5ar1 1 1 Dtx3l Sesn1 Cd93 0 0 Fcgr4 Sh3bp5 B4galt6 0 Ifi204 Wwp1 Gas6 -1 -1 Ifit2 C5ar1 -1 Maf Rnf213 Sqstm1 Serpinb6a Gbp4 Xylt2 Hpgds Iigp1 Hmox1 Igfbp4 Nampt Cdk18 Cxcl9 Coro1a Fcgr1 Sec14l1 Lgmn Ifit1 Rasgrp3 Pou2f2 Ifi211 Zmiz1 Sema4d Irgm2 Osbpl8 Cybb Csf2rb Stk17b Cxcr4 Gbp7 Cxcr4 Mpeg1 Fcgr3 Mafg Marcksl1 Parp14 Hpgd Ifitm2 Slfn5 Rgs10 Selenop Sp110 Lhfpl2 Hpgd Batf2 Fmnl1 Clec4d Igtp Irf2bp2 Fgl2 Clec12a Gbp2 Cx3cr1 Cd14 Ifi47 Ldlrap1 Pid1 Gbp3 Adam8 Lst1 Isg15 Dhrs3 Itga6 Stat1 Cd300lb Rab32 Irgm1 Rps28 Cxcl16 Trafd1 Rps21 Rtp4 Mndal Rps29 Cx3cr1 Trim30a Slbp Tmsb4x Ifi203 Rpl38 Ifitm3 Zyx Gmnn Wfdc17 St3gal5

Ch25h S S Ywhah G1 G1

Cd274 G2/M G2/M Lsp1 Rnf19b Ifi27l2a Ccl2 Spp1 Ccl12 M0 M1 Sod1 Apobec3 (CTR) (IFNG) Pf4 Pnp Arsb S S S S G1 G1 G1 G1 G2/M G2/M G2/M G2/M

M0 M1 M0 M2 (CTR) (IFNG) (CTR) (IL-4) F G Cxcl10 Gbp5 Irf1 Fam26f Ptpre Prkcd Gatm Cblb Arg1 G1 0-520 0-520 0-520 0-520 G1 0-520 0-520 0-520 0-150 0-150 S S CTR CTR G2/M G2/M

G1 G1 4 S - S IL IFNG G2/M G2/M Supplementary Figure 5.

A B C Priming trajectory Cis-regulatory regions (CREs) Marker gene scores “Transient” M0(CTR) CRE biased - n=88 C3 biased - n=233 C1 biased - “Primed” n=28 CREs C4 biased

M2(IL-4) biased - - n=21 n=102 C1 C2 biased - n=85

M2p(pIL-4) C3 biased - C1 C2 C5 C6 C3 C4 n=85

UMAP2 C3 C4 C5 C6 C1 C2 C2 biased n=8 UMAP1 Pseudotime order - C4

START END M2 M0 M2p M2p M0 M2 (IL-4) (CTR) (pIL-4) (pIL-4) (CTR) (IL-4) Z-scores Gene score

-2 2 -1.8 2 D C2-biased gene scores

Col6a3 1 9430019J16Rik 0 Top2a

Fam71f1 -1 Msx3 Csmd2 Mxd3 Clec10a Gm839 Faap20 Gm20619 Dbx2 Mir7661 Dhrs9 Ang6 2210417A02Rik Tubb5 Mdc1 Ang2 Cavin2 Slc36a3 Snap47 Caly Gatm Spint1 Frmpd3 Fermt2 Siglec15 Rnase2a Slc9a6 C1 C2 C3 C4 C5 C6

Gene score -1 0 1 Supplementary Figure 6.

A Chromatin accessibility (Gene scores)

Cxcl9 Irf8 Cxcl10 Mki67

Log2(NC+1) UMAP2 Min Max UMAP1 Cxcl9 Irf8 Cxcl10 Mki67 3.0

0.9 2.0 1.4 2.5 1.2 0.6 1.5 2.0 1.0 1.0 Genescore 0.3 1.5 0.8 0.5 0.6 C1 C2 C3 C4 C5 C6 C7 C8 C1 C2 C3 C4 C5 C6 C7 C8 C1 C2 C3 C4 C5 C6 C7 C8 C1 C2 C3 C4 C5 C6 C7 C8 B

Gene expression (Gene integration scores)

Cxcl9 Irf8 Cxcl10 Mki67

#Counts

UMAP2 Min Max UMAP1 Cxcl9 Irf8 Cxcl10 Mki67 12 20 5

9 15 60 4 3 6 10 40 2 3 5 20 1

Geneintegration score 0 0 0 0 C1 C2 C3 C4 C5 C6 C7 C8 C1 C2 C3 C4 C5 C6 C7 C8 C1 C2 C3 C4 C5 C6 C7 C8 C1 C2 C3 C4 C5 C6 C7 C8 C Cell cycle distribution * M0(CTR) M2(IL-4) M2p(pIL-4) 80 11 * 10 72.8 72.3 70.0 60 9

40 8 Count 7 12.4 12.4 13.5 20 6.56 9.16 9.72 6 Percent of cells in cell cycle 0 Macrophages in G2/M (%) 5 S S S G1 G1 G1 IL-4 G2/M G2/M G2/M CTR pIL-4 VybrantDyeCycle CTR IL-4 pIL-4 Supplementary Figure 7.

A B C C0 G1 4000 3655 n=5997 C1 S M0(CTR) 3000 C2 G2/M C3 2000 Sensitive Insensitive 1586 n=3776 n=7327 C4

Macrophage number 1000 537 C5 134 68 2 2 15

0 UMAP2 C6 UMAP2 0 0.5 2 3.5 5 10 21 Z

- Post injury (days) UMAP1 UMAP1 scores D E C0 C1 -2 C2 C3 C4 UMAP2 UMAP1

Cellcycleinsensitive inCTR(n=7327) F Mrc1 Msr1 Itgam Adgre1 3 3 3 3

2 2 2 2 G1 S G2/M

1 1 1 1 Expressionlevel

0 0 0 0 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 M0(CTR) G H

Mcm6 3 Pcna Mki67 Cenpa Fn1 5 Mmp9 Col1a1 Col1a2 Acta2 4 3 3 4 4 3 2 2 2 3 3 2 3 2 2 2 2 2 1 1 1 1 1 1 1 1

Expressionlevel 1 Expressionlevel

0 0 0 0 0 0 0 0 0 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4 C0 C1 C2 C3 C4

I J Stented wound healing model Collect tissue slice H&E stain

10x Visium

Spatial gene expression Cluster1 Cluster2 Cluster3 Cluster4 Cluster5 Cluster6 Cluster7

Uninjured 7 days after operation Log

10 2 expression

Col1a2 0

7

Mmp9 0

5

Vcam1 0