Thi o IL-4c - NatureSupplementary Figure 1 Monocyte -derived inflammatory macrophages adopt a tissue -resident phenotype after long-term residency in the peritoneal cavity. (a) Schematic

Supplementary Figure 1

Monocyte-derived inflammatory macrophages adopt a tissue-resident phenotype after long-term residency in the peritoneal cavity.

(a) Schematic of short-term transfer of monocyte-derived inflammatory macrophages from thioglycollate treated CD45.1 donor mice into CD45.2 recipient mice, rested for 24hrs and subsequently treated with IL-4c. (b) Quantification of mean fluorescence intensity values of PD-L2 and CD206 expression on donor and recipient macrophages. (c and d) Flow cytometric analysis of adoptive cell transfer of macrophages flow sorted from (c) thioglycollate-treated mice or (d) IL-4c treated mice and transferred into CD45.2 recipient mice and

then treated with IL-4c 24hrs after transfer. Representative histograms display surface expression of F4/80, CD206, PD-L2 and MHCII in Donor CD45.1

+ CD45.2

- (Black line) and recipient CD45.1

-, CD45.2

+ (Grey shaded) CD11b

+, F4/80

+ macrophages. (e) IL-4c induced

tissue resident M2 macrophages rapidly disappear after transfer into an inflammatory environment. CD11b+, F4/80

+ macrophages

sorted from CD45.1 donor mice treated with IL-4c were adoptively transferred into CD45.2 recipient mice, 24hrs later the recipient mice were treated with thioglycollate + IL-4c prior to analysis.

Figure S1

Thio

CD45.1 CD45.2

24hrs

+/- IL-4c

45.1

45.2

45.1

45.2

101

102

103

104

D R R D

!IL!4%+IL!4%

***

IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio+IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio

CD45.1 CD45.2

24hrs+/- IL-4c

F4/80 CD206 PD-L2 MHCII

CD45.2 Recipient

CD45.1 Donor

45.1

45.2

45.1

45.2

102

103

104

D R R D

!IL!4%+IL!4%

MeanFluorescence

CD206PD-L2

Donor

-IL-4c

+IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

-IL-4c

+IL-4c

F4/80 CD206 PD-L2 MHC II

a b

c d

ns***

Figure2

<Alexa Fluor 488-A>: CD451



: F

48

0

25.1

1.51




: F

48

0

53.5

0.0213

IL-4c

CD45.1 CD45.2

24hrs+/- Thio+IL-4c

CD45.1

F4

/80

Thio+IL-4

cContro

l

e

45.1

45.2

45.1

45.2

101

102

103

104

D R R D

!IL!4%+IL!4%

***

IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio+IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio

CD45.1 CD45.2

24hrs+/- IL-4c


CD45.2 Recipient

CD45.1 Donor

45.1

45.2

45.1

45.2

102

103

104

D R R D

!IL!4%+IL!4%

MeanFluorescence

CD206PD-L2

Donor

-IL-4c

+IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

-IL-4c

+IL-4c


a b

c d

ns***

Figure2




: F

48

0

25.1

1.51


: F

48

0

53.5

0.0213

IL-4c

CD45.1 CD45.2

24hrs+/- Thio+IL-4c

CD45.1

F4

/80

Thio+IL-4

cContro

l

e

a b

IL-4c

CD45.1 CD45.2

24hrs

+/- Thio+IL-4c Thio

+IL

-4c

Contro

l

e

CD45.1

F4/8

0-

IL-4

c+IL

-4c

F4/80 CD206 PD-L2103 104 1051020103 104 1051020103 1040 105

0

20

40

60

80

100

% o

f M

ax

d

45.1

45.2

45.1

45.2

101

102

103

104

D R R D

!IL!4%+IL!4%

***

IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio+IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio

CD45.1 CD45.2

24hrs+/- IL-4c


CD45.2 Recipient

CD45.1 Donor

45.1

45.2

45.1

45.2

102

103

104

D R R D

!IL!4%+IL!4%

MeanFluorescence

CD206PD-L2

Donor

-IL-4c

+IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

-IL-4c

+IL-4c


a b

c d

ns***

Figure2




: F

48

0

25.1

1.51


:

F4

80

53.5

0.0213

IL-4c

CD45.1 CD45.2

24hrs+/- Thio+IL-4c

CD45.1

F4

/80

Thio+IL-4

cContro

l

e

Thio+IL-4c

CD45.1 CD45.2

24hrs

+/- IL-4c

c

IL-4c

CD45.1 CD45.2

24hrs

+/- IL-4c

45.1

45.2

45.1

45.2

101

102

103

104

D R R D

!IL!4%+IL!4%

***

IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio+IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio

CD45.1 CD45.2

24hrs+/- IL-4c


CD45.2 Recipient

CD45.1 Donor

45.1

45.2

45.1

45.2

102

103

104

D R R D

!IL!4%+IL!4%

MeanFluorescence

CD206PD-L2

Donor

-IL-4c

+IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

-IL-4c

+IL-4c


a b

c d

ns***

Figure2




: F

48

0

25.1

1.51


:

F4

80

53.5

0.0213

IL-4c

CD45.1 CD45.2

24hrs+/- Thio+IL-4c

CD45.1

F4

/80

Thio+IL-4

cContro

l

e

45.1

45.2

45.1

45.2

101

102

103

104

D R R D

!IL!4%+IL!4%

***

IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio+IL-4c

CD45.1 CD45.2

24hrs+/- IL-4c

Thio

CD45.1 CD45.2

24hrs+/- IL-4c


CD45.2 Recipient

CD45.1 Donor

45.1

45.2

45.1

45.2

102

103

104

D R R D

!IL!4%+IL!4%

MeanFluorescence

CD206PD-L2

Donor

-IL-4c

+IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

-IL-4c

+IL-4c


a b

c d

ns***

Figure2


:

F4

80

25.1

1.51




: F

48

0

53.5

0.0213

IL-4c

CD45.1 CD45.2

24hrs+/- Thio+IL-4c

CD45.1

F4

/80

Thio+IL-4

cContro

l

e

-IL

-4c

+IL

-4c


0

20

40

60

80

100

% o

f M

ax

103 1040 105 103 104 1051020103 104 1051020 103 1040 105102

-IL

-4c

+IL

-4c

F4/80 CD206 PD-L2 MHCII103 104 1051020 103 1040 105 103 104 1051020 103 1040 105

0

20

40

60

80

100

% o

f M

ax

0102

103

104

105

010

2

103

104

105

53.5

0.0213

25.1

1.51

PD-L2 CD206

Nature Immunology: doi:10.1038/ni.3734

1wk 4wks 8wks0

50

100

% o

f C

D11b+

td

Tom

ato

+

F480- CD206-F480- CD206+F480+ CD206+F480+ CD206-

<Alexa Fluor 488-A>: CD45-1



: F

48

0

72.4

0.252




: F

48

0

6.52

0.0568




: F

48

0

69.6

0.0565

+IL-4c-IL-4cThio+IL-4




: F

48

0

14.9

2.1e-3

CD45.1

F4

/80

Thio-IL-4c

<PE-Cy7-A>: F480

:

ED

U

9.73


:

ED

U

16.7 0.185

1.9881.2


:

ED

U

17.3 0.0283

0.24782.4 <PE-Cy7-A>: F480

:

ED

U

7.82<PE-Cy7-A>: F480

:

ED

U

20.8

<PE-Cy7-A>: F480

:

ED

U

21.5

CD45.1E

dU

Thio+IL-4

Thio

F4/80

Ed

U

DonCD45.1RecCD45.2

24hrsposttran

sfer

Thio+IL-4

Thio

d e

b

ThioThio+IL-4c

CD45.1 CD45.2

8wks+/- IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

RecipientDonor

i

<PE-Cy7-A>: F480

:

PD

L2

<PE-Cy7-A>: F480<

PE

-A>

: P

DL2

<PE-Cy7-A>: F480

<A

PC

-A>

: m

r1

<PE-Cy7-A>: F480

<A

PC

-A>

: m

r1<PE Cy7-A>: F480

:

PD

L2

92.3 4.12

0.1683.38

<PE Cy7-A>: F480

<A

PC

-A>

: M

R1

83.5 5.56

0.12410.8

+IL-4c-IL-4cThio+IL-4c

F4/80C

D2

06

F4/80

PD

-L2

h

CD45.1

Donor%

Recipient%

CD

45

.2

a

1wkThio+IL-4c

4wks 8wks

93.7 4.24

0.03562.06

28.7 56.1

13.21.98

10.9 19.1

67.92.15

F4/80

CD

20

6

8wks4wks1wk

<PE-Cy7-A>: F480

:

Ed

U

20.9

<PE-Cy7-A>: F480

:

Ed

U

17.5<PE-Cy7-A>: F480

:

Ed

U

23.5


:

Ed

U

16.1 0.0128

0.04683.8


:

Ed

U

12.8 0.0321

0.14887

CD45.1

Ed

U

<PE-Cy7-A>: F480

:

Ed

U

21.1

F4/80

Ed

U

8wksposttran

sfer

f g

Thio+IL-4

Thio

DonCD45.1RecCD45.2

+IL-4c

Figure3

c

CD45.1

CD

45

.28wksposttransferPretransfer

1wk 4wks 8wks0

50

100

% o

f C

D11b+

td

Tom

ato

+

F480- CD206-F480- CD206+F480+ CD206+F480+ CD206-




: F

48

0

72.4

0.252


:

F4

80

6.52

0.0568




: F

48

0

69.6

0.0565





: F

48

0

14.9

2.1e-3

CD45.1

F4

/80

Thio-IL-4c

<PE-Cy7-A>: F480

:

ED

U

9.73


:

ED

U

16.7 0.185

1.9881.2


: E

DU

17.3 0.0283

0.24782.4 <PE-Cy7-A>: F480

:

ED

U

7.82<PE-Cy7-A>: F480

:

ED

U

20.8

<PE-Cy7-A>: F480

:

ED

U

21.5

CD45.1

Ed

UThio+IL-4

Thio

F4/80

Ed

U

DonCD45.1RecCD45.2

24hrsposttran

sfer

Thio+IL-4

Thio

d e

b

ThioThio+IL-4c

CD45.1 CD45.2

8wks+/- IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

RecipientDonor

i

<PE-Cy7-A>: F480

:

PD

L2

<PE-Cy7-A>: F480

: P

DL2

<PE-Cy7-A>: F480

<A

PC

-A>

: m

r1

<PE-Cy7-A>: F480

<A

PC

-A>

: m

r1

<PE Cy7-A>: F480

: P

DL

2

92.3 4.12

0.1683.38

<PE Cy7-A>: F480

<A

PC

-A>

: M

R1

83.5 5.56

0.12410.8


F4/80

CD

20

6

F4/80

PD

-L2

h

CD45.1

Donor%

Recipient%

CD

45

.2

a

1wkThio+IL-4c

4wks 8wks

93.7 4.24

0.03562.06

28.7 56.1

13.21.98

10.9 19.1

67.92.15

F4/80

CD

20

6

8wks4wks1wk

<PE-Cy7-A>: F480

:

Ed

U

20.9

<PE-Cy7-A>: F480

:

Ed

U

17.5<PE-Cy7-A>: F480

:

Ed

U

23.5


:

Ed

U

16.1 0.0128

0.04683.8


:

Ed

U

12.8 0.0321

0.14887

CD45.1

Ed

U

<PE-Cy7-A>: F480

:

EdU

21.1

F4/80

Ed

U

8wksposttran

sfer

f g

Thio+IL-4

Thio

DonCD45.1RecCD45.2

+IL-4c

Figure3

c

CD45.1

CD

45

.2

8wksposttransferPretransfer

a

b Thio Thio+IL-4c

d

e

Thio+IL-4c

CD45.1 CD45.2

8wks

+/- IL-4c

Thio+IL-4c

1wk 4wks 8wks

1wk 4wks 8wks0

50

100

% o

f C

D11b+

td

Tom

ato

+

F480- CD206-F480- CD206+F480+ CD206+F480+ CD206-




: F

48

0

72.4

0.252




: F

48

0

6.52

0.0568




: F

48

0

69.6

0.0565





: F

48

0

14.9

2.1e-3

CD45.1

F4

/80

Thio-IL-4c

<PE-Cy7-A>: F480

:

ED

U

9.73


:

ED

U

16.7 0.185

1.9881.2


:

ED

U

17.3 0.0283

0.24782.4 <PE-Cy7-A>: F480

:

ED

U

7.82<PE-Cy7-A>: F480

:

ED

U

20.8

<PE-Cy7-A>: F480

:

ED

U

21.5

CD45.1

Ed

UThio+IL-4

Thio

F4/80

Ed

U

DonCD45.1RecCD45.2

24hrsposttran

sfer

Thio+IL-4

Thio

d e

b

ThioThio+IL-4c

CD45.1 CD45.2

8wks+/- IL-4c

F4/80 CD206 PD-L2

-IL-4c

+IL-4c

RecipientDonor

i

<PE-Cy7-A>: F480

:

PD

L2

<PE-Cy7-A>: F480

: P

DL

2

<PE-Cy7-A>: F480

<A

PC

-A>

: m

r1

<PE-Cy7-A>: F480

<A

PC

-A>

: m

r1

<PE Cy7-A>: F480

:

PD

L2

92.3 4.12

0.1683.38

<PE Cy7-A>: F480

<A

PC

-A>

: M

R1

83.5 5.56

0.12410.8


F4/80C

D2

06

F4/80

PD

-L2

h

CD45.1

Donor%

Recipient%

CD

45

.2

a

1wkThio+IL-4c

4wks 8wks

93.7 4.24

0.03562.06

28.7 56.1

13.21.98

10.9 19.1

67.92.15

F4/80

CD

20

6

8wks4wks1wk

<PE-Cy7-A>: F480

:

Ed

U

20.9

<PE-Cy7-A>: F480

:

Ed

U

17.5<PE-Cy7-A>: F480

:

Ed

U

23.5


:

Ed

U

16.1 0.0128

0.04683.8


:

Ed

U

12.8 0.0321

0.14887

CD45.1

Ed

U

<PE-Cy7-A>: F480

:

Ed

U

21.1

F4/80

Ed

U

8wksposttran

sfer

f g

Thio+IL-4

Thio

DonCD45.1RecCD45.2

+IL-4c

Figure3

c

CD45.1

CD

45

.28wksposttransferPretransfer

Thio+IL-4c

Pretransfer8wks post transfer

-IL-4c +IL-4c

Figure S2

0102

103

104

105

010

2

103

104

105 94 4.1

0.0452.1

F4/80

CD

206

29 56

142

13 10

688.9

CD45.1

F4/8

0F4/80

PD

-L2

CD

206

CD45.1

CD

45.2

0102

103

104

105

010

2

103

104

105 70

0.057

72

0.25

6.5

0.057

8.2

2.3e-3

103 104 1051020

10

31

04

10

50

0102

103

104

105

0

103

104

105

0102

103

104

105

0

103

104

105

Thio

Stat6-/-

CD45.2

WT

CD45.1

24hrs

+/- IL-4c

Ed

U

Ed

U

CD45.2

Ed

U

F4/80

Ed

U

Thio

Sta t6 -/-

CD45.2WT

CD45.1

24hrs+/- IL-4c


CD45.1 Recipient

CD45.2 Donor

-IL-4c

+IL-4c

-IL-4c

+IL-4c

a

c

Figure7

b0.137 5.47e-3

2.4597.4

16.4 0.0102

1.0282.5

0.109

17

0.221

0.763

DonCD45.2RecCD45.1

-IL-4c

Thio

WTCD45.1

24hrs+/- IL-4c

CD45.2 Recipient

CD45.1 Donor

Sta t6 -/-

CD45.2


0.0594 0.0171

2.497.5

0.477 0.0488

1.9197.6

0.113

0.392

0.719

2.56

CD45.1 F4/80

d DonCD45.1RecCD45.2

-IL-4c

+IL-4c

+IL-4c

CD45.2 Donor

Thio

Sta t6 -/-

CD45.2WT

CD45.1

8wks+/- IL-4c

eCD45.1 Recipient

-IL-4c

+IL-4c

F4/80 CD206 PD-L2

99.80.0105

-IL-4c

+IL-4c

CD

45

.2

CD45.1

99.96.97e-3

CD

45

.2

CD45.1

99.11.28e-3

No transfer

8wks+ IL-4c

CD45.2 Donor

Thio

Ir f4 -/-

CD45.2WT

CD45.1

fCD45.1 Recipient

+IL-4c

F4/80 CD206 PD-L2

+IL-4c

CD

45

.2

CD45.10 103

104

105

0

103

104

105


:

CD

45

2

98.5

0.0209

0 102

103

104

105

<PE-Cy7-A>: F480

0

20

40

60

80

100

% o

f M

ax

0102

103

104

105

<APC-A>: MR1

0

20

40

60

80

100

% o

f M

ax

0 103

104

105

<PE-A>: PDL2

0

20

40

60

80

100

% o

f M

ax


-IL-4

c+

IL-4

c%

of M

ax

0 103

104

105

0

20

40

60

80

100

1wk 4wks 8wks0

25

50

75

100

% o

f C

D11b

+

F480- CD206-F480- CD206+F480+ CD206+F480+ CD206-

CD45.2 F4/80

EdU

0102

103

104

105

0

102

103

104

105 16 0.76

0 103

104

105

0

102

103

104

105 11 8.3e-3

0.8788

0.057 2.5e-3

1.499

0.46 0.25

Recipient

CD45.1

Donor

CD45.2

EdU

-IL-4

c+

IL-4

c

1wk 4wk 8wk

c 24hrs 8wks

Thio

Thio

+ IL-4

c

CD45.1

EdU

16 0.013

0.04684

13 0.032

0.1587

17 0.19

281

0102

103

104

105

010

2

103

104

105 17 0.028

0.2582

F4/80

-IL-4c

+IL-4c

-IL-4c

+IL-4c



Fate-mapping of peritoneal macrophages after thioglycollate injection.

(a) Schematic of timecourse analysis of peritoneal macrophages 1 week (n=4), 4 weeks (n=4) or 8 weeks (n=4) after thioglycollate injection. Stacked bar graph showing the relative proportion of F4/80 and/or CD206 expression. (b) Representative FACS plots are

shown displaying the frequency of CD45.1+ (blue) donor cells in CD45.2 (grey) recipient mice treated with or without IL-4c after 8 weeks

of residency. Flow cytometry analysis of transferred cells shows acquisition of tissue resident phenotype by CD45.1 donor cells in the presence or absence of IL-4c treatment. IL-4c treatment increases the frequency of CD45.1

+ F4/80

+ cells. (c) Representative FACS

plots showing the frequency of EdU+ cells in recipient and donor populations in response to IL-4c given after transfer and resting for

24hrs for short term residency or long term residency for 8 weeks. (d) Schematic of adoptive transfer of Thio-elicited monocyte-derived macrophages from Stat6

-/- CD45.2 donor mice transferred into WT CD45.1 recipient mice rested for 24hrs and then treated with IL-4c.

Histograms display expression of F4/80, CD206, PD-L2 and MHCII in donor CD45.2+ CD45.1

- (Black) and recipient CD45.2

-, CD45.1

+

(Grey shaded) CD11b+ cells. (e) Representative FACS plots showing the frequency of EdU

+ cells in WT recipient and Stat6

-/- donor

CD11b+ F4/80

+ macrophages in response to IL-4c given after resting for 24hrs.



Transcriptional and chromatin landscape reprogramming during macrophage conversion.

(a) Pairwise Pearson’s correlation analysis and (b) PCA of transcriptional profiles in AAMmono

, AAMconv

and AAMres

. (c) Heatmap visualizing the expression values of 6 specific genes across the different populations of macrophages. (d) Top 10 GO terms enriched in

the 3966 genes upregulated in AAMmono

when compared to AAMconv

(top) and top 10 GO terms enriched in the 675 genes upregulated in AAM

conv when compared to AAM

res (bottom). (e) Pairwise Pearson’s correlation analysis and (f) PCA of accessible chromatin regions

in AAMmono

, AAMconv

and AAMres

. On PCA plots, red circles represent AAMmono

, orange squares represent AAMconv

and blue triangles represent AAM

res in PCA plots.

0 10 20 30 40 50

intracellular signal transduction

immune system process

localization

primary metabolic process

organic substance metabolic process

macromolecule metabolic process

positive regulation of biological process

cellular macromolecule metabolic process

cellular metabolic process

metabolic process

Transform of Tissue_conv_vs_Mono_down

-log10 P value

0 10 20 30 40

cellular nitrogen compound metabolic process

DNA replication

organelle organization

DNA metabolic process

metabolic process

primary metabolic process

cellular metabolic process

organic substance metabolic process

cell cycle process

cell cycle

Transform of Tissue_conv_vs_Tissue_up

-log10 P value

> in AAMmono vs. AAMconv (N=3966)

> in AAMconv vs. AAMres (N=675)

−2 0 0

−1 0 0

0

1 0 0

2 0 0

−2 0 0 −1 0 0 0 1 0 0 2 0 0

PC1: 44% v ariance

PC

2:

14

% v

ari

an

ce

Population

M ono_1wk

Tissue

Tissue_Con v

b

f

0

100

200

-200

-100

0-200 -100 200100

PC1: 44% variance

AAMmono

AAMres

AAMconv

PC

2: 14%

variance

−2 0 0

−1 0 0

0

1 0 0

2 0 0

−2 0 0 −1 0 0 0 1 0 0 2 0 0

P C 1: 28% v a r iance

PC

2:

17

% v

ari

an

ce

Popu la tion

M ono_1w k

T issue

T issue_C on v

0

100

200

-200

-100

0-200 -100 200100

PC1: 28% variance

AAMmono

AAMres

AAMconv

PC

2: 17%

variance

d

aM

on

o

Conv

Conv

Conv

Mono

Mono

Mono

Res

Res

Res

Re

s

Re

s

Re

s

Co

nv

Co

nv

Co

nv

Mo

no

Mo

no

Mo

no

_1

wk

_1

Mo

no

_1

wk

_3

Mo

no

_1

wk

_2

Tis

su

e_

Co

nv

_2

Tis

su

e_

Co

nv

_1

Tis

su

e_

Co

nv

_3

Tis

su

e_

2

Tis

su

e_

3

Tis

su

e_

1

M ono_1w k_1

M ono_1w k_3

M ono_1w k_2

T issue_C on v_2

T issue_C on v_1

T issue_C on v_3

T issue_2

T issue_3

T issue_1

0 .9 6 0 .9 8 1

Va lu e

Color Key

Mono_1wk_1Mono_1wk_2Mono_1wk_3

Tissue_1Tissue_2Tissue_3

Tissue_Conv_1

Tissue_Conv_2

Tissue_Conv_3

Tissue_Conv_3

Tissue_Conv_2

Tissue_Conv_1

Tissue_3

Tissue_2

Tissue_1

Mono_1wk_3

Mono_1wk_2

Mono_1wk_1

04080

Value

Color Key

Pearson’s Correlation

0.96 1RNAseq

e

Conv

Conv

Mono

Res

Mono

Mono

Res

Res

Conv

Mo

no

Mo

no

Mo

no

Re

s

Re

s

Co

nv

Re

s

Co

nv

Co

nv

Mo

no

_1

wk

_1

Mo

no

_1

wk

_2

Mo

no

_1

wk

_3

Tis

su

e_

3

Tis

su

e_

2

Tis

su

e_

Co

nv

_1

Tis

su

e_

1

Tis

su

e_

Co

nv

_2

Tis

su

e_

Co

nv

_3

M ono_1w k_1

M ono_1w k_2

M ono_1w k_3

T issue_3

T issue_2

T issue_C on v_1

T issue_1

T issue_C on v_2

T issue_C on v_3

0 .85 0 .9 0 .95 1

Va lue

Color Key

Mono_1wk_1Mono_1wk_2Mono_1wk_3

Tissue_1Tissue_2Tissue_3

Tissue_Conv_1

Tissue_Conv_2

Tissue_Conv_3

Tissue_Conv_3

Tissue_Conv_2

Tissue_Conv_1

Tissue_3

Tissue_2

Tissue_1

Mono_1wk_3

Mono_1wk_2

Mono_1wk_1

04080

Value

Color Key

Pearson’s Correlation

0.85 1

ATACseq

c

Mrc1

Ccr2

Pdcd1lg2

Ucp1

Gata6

Aldh1a2

Tis

su

e_

Co

nv

Tis

su

e_

Co

nv

Tis

su

e_

Co

nv

Tis

su

e

Tis

su

e

Tis

su

e

Mo

no

_1

wk

Mo

no

_1

wk

Mo

no

_1

wk

G ata6

U cp1

P dcd1 lg2

C cr2

M rc1

A ldh1a2

−1 .5 0 1

Va lue

Color Key

Co

nv

Co

nv

Co

nv

Re

s

Re

s

Re

s

Mo

no

Mo

no

Mo

no

-2 2

rlog read counts

Figure S3



Baseline disruption of peritoneal tissue-resident macrophages in vitamin A–deficient mice.

(a) Total number of cells collected from the peritoneal cavity of vitamin A deficient (Vit-ADEF

) or control (Vit-ACON

) mice via peritoneal lavage. (b,c) Total number of F4/80

hi CD206

- (b) or F4/80

int CD206

+ (c) cells in the peritoneal cavity in Vit-A

DEF or Vit-A

CON mice. **P <

0.01. Unpaired Students T-test.

Vit A C

trl

Vit A D

ef

0.0

5.0×1004

1.0×1005

1.5×1005

2.0×1005

2.5×1005

**

Vit A C

trl

Vit A D

ef

0.0

5.0×1004

1.0×1005

1.5×1005

2.0×1005

2.5×1005**

Vit A C

trl

Vit A D

ef

0

2×1005

4×1005

6×1005

8×1005

**

a b c

Tota

l num

ber

of cells

Tota

l num

ber

of

F4/8

0h

iC

D206

-cells

Tota

l num

ber

of

F4/8

0in

tC

D206

+cells

Figure S4


a b

c 50μm

50μm

50μm

50μm50μm50μm

50μm50μm50μm

50μm50μm50μm

6wks PI 6wks PI

8wks PI 8wks PI 8wks PI

12wks PI 12wks PI

6wks PI

12wks PI

Immature

Immature

Mature

Mature

Immature

EdU UCP1

50μm 50μm50μm

50μm 50μm50μm

500μm

Mature

Immature

Mature

ImmatureEdU UCP1 EdU

UCP1

EdUUCP1

8 weeks post-infection EdUUCP1DAPI

EdUDAPI

Figure S5

Uni

nfec

t ed

9 wee

ks

12 w

eeks

0.000

0.001

0.002

0.003

0.004

0.005

UCP1

Gene e

xpre

ssio

n (

a.u

.)



Expression of UCP1 in S. mansoni infection associated with EdU localization in mature liver granulomas.

(a) Transcript expression of Ucp1 in whole liver from mice infected with S. mansoni at 9 weeks and 12 weeks post infection. (b)

Representative immunofluorescence images of S. mansoni-infected liver granulomas at different timepoints post infection stained with DAPI (blue) and Click-it EdU (red) taken from mice pulsed with EdU 3 hours prior to sacrifice. Eggs are outlined in white. (c) Slide-scanned, immunofluorescence image of S. mansoni-infected liver granulomas taken at 8 weeks post infection and stained with DAPI (blue), anti-UCP1 (green) and Click-it EdU (red) in vitamin A control mice pulsed with EdU 3 hours prior to sacrifice. Scale bars represent 50 microns or 500 microns as indicated.



STAT6 regulates phenotypic conversion of peritoneal AAMmono

cells after S. mansoni egg injection.

(a) Schematic of S. mansoni egg injection in Cx3cr1creERT2-IRESYFP/+

Rosa26stopfl-tdTomato/+

vitamin A deficient (Vit-ADEF

) or control (Vit-

Vit-ACON

Vit-ACON Vit-ADEF

CD11c

CD

11b

Lungs

Vit-ADEF

Cx3cr1creER-YFP/+

R26tdTomato/+

tdTomato

SS

C-A

14d 7d

tdTomato

CX

3C

R1-Y

FP

a

Figure S6

0 1 2 3 4 5 6 7 80

20

40

60

80

100

Weeks post-infection

Perc

ent su

rviv

al

S. mansoni

Stat6-/- Inf (5)

Stat6-/- Uninf (2)

2 3 4 5 6 716

18

20

22

24


Tota

l body w

eig

ht (g

)

Stat6-/- Inf

Stat6-/- Uninf

2 3 4 5 6 716

18

20

22

24


Tota

l bod

y w

eig

ht (g

)

Stat6-/- Inf

Stat6-/- Uninf

STAT6-/-:WTWT:WT

Mixed BM Chimera

STAT6-/- (CD45.2)WT (CD45.1)

NoInfection

b

c

0102

103

104

105

010

2

103

104

105 8.1 2.9

0.8988

23 3.2

0.5874

0102

103

104

105

010

2

103

104

105

5.3 5.2

24 11

7.258

0102

103

104

105

010

2

103

104

105 23 11

4.762

STAT6-/-:WTLungs

WT:WT

Mixed BM Chimera

STAT6-/- (CD45.2)WT (CD45.1)

14d 7d

NoEggs

CD

206

MH

CII

PD

-L2

F4/80

EdU

WT(CD45.1) Stat6-/-(CD45.2)

No Eggs EggsWT(CD45.1) Stat6-/-(CD45.2)

WT:WT WT:Stat6-/-

No E

ggs

Eggs

CD45.2

CD

45.1

0102

103

104

105

010

2

103

104

105

57

22

33

39

27

37

30

32

0102

103

104

105

010

2

103

104

105

0.28 0.29

3.1 2.1

9.3 37

2924

1.7 39

4415

18 47

2015

9.5 58

257.2

11 29

3823

1.6 7.7

7615

0.48 0.67

1485

1.1 2.6

2374

31 21

9.439

20 7.8

1062

0.3 0.37

3268

0.064 0.19

2080

Infected

Uninfected

Vit-ACON Vit-ADEF


ACON

) mice. Representative flow cytometry plots of fate-mapped monocyte-derived macrophages in the lung after S. mansoni egg challenge in the lung. (b) Schematic of S. mansoni egg injection in WT:WT (n=5) or Stat6

-/-:WT (n=5) mixed bone marrow chimeric mice

whereby mice were sensitized with eggs via i.p. injection then challenged i.v. with eggs after 2 weeks. PECs and lung macrophages were analyzed after 8 days of rest and pulsed with EdU 3hrs prior to sacrifice. Representative flow cytometry plots display macrophage phenotypes from the peritoneal cavity and lung in mixed bone marrow chimeric mice treated with or without eggs. (c) Schematic of S. mansoni infection in mixed bone marrow chimera Stat6

-/-:WT mice. Cumulative body weight during the infection reveals loss of weight

after 5 weeks post-infection. Kaplan-Meyer survival curve displays rapid mortality of Stat6-/-

:WT chimeric mice at 7 weeks post-infection.

Documents

Thi o IL-4c - NatureSupplementary Figure 1 Monocyte -derived inflammatory macrophages adopt a tissue -resident phenotype after long-term residency in the peritoneal cavity. (a) Schematic