0 = Normal

1 = Minimal

2 = Mild

3 = Moderate

4 = Moderate

to Marked

5 = Marked

Figure S1. Glomerular pathology and glomerular filtration rate of Tnip1-/- mice

(A) Correlation of glomerular area and pathology score assessing GN in Tnip1-/- mice.

Histology-based kidney pathology of Tnip1-/- mice (various age groups) was assessed by

GN score and glomerular area measurements (detailed in Methods section), and is

displayed as correlation analysis. Pearson r = Pearson correlation coefficient. (B) Kidney

pathology as assessed by GN score of Tnip1+/+ and Tnip1-/- mice during time. (C) H&E-

stained kidney sections of Tnip1-/- mice showing examples of glomerular lesions with

different grades used for scoring (see Methods section). Original magnification 400x.

5 -8 9 -1 2 1 3 -1 6 > 1 6

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

GN

(S

core

)

****** ****

5-8

9-1

2

13-1

6>16

0

1

2

3

4

5

p r o te in u r ia

wt

T n ip 1 - /-

Tnip1+/+

Tnip1-/-

B

C

Rela

tive G

FR

(%

)

0

5 0

1 0 0

Tnip1-/-

F

**

A

0 2 0 4 0 6 0 8 0 1 0 0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

Pathology Score

Pearson r = 0.81G

lom

eru

lus a

rea (

µm

2)

0

2 0

4 0

6 0

Ki6

7+

cells

(num

ber/

glo

meru

lus)

Tnip1+/+ Tnip1-/-

0

2 0

4 0

6 0

CD

45

+cells

(num

ber/

glo

meru

lus)

Tnip1+/+ Tnip1-/-

********

Ki67 DESMIN CD45 merge

0

5 0

1 0 0

1 5 0

Ki6

7+

cells

(% o

f to

tal cells

)

****

CD45+

KI67+

DESMIN+

KI67+

D

E

Figure S1 (continued)

(D) Multicolor immunofluorescence analysis of kidney tissue sections from16 week-old

Tnip1-/- mice using antibodies against Ki67, DESMIN and CD45. Scale bars 10 µm. (E)

Quantitative analysis of multicolor immunofluorescence images (as shown in (E)) of 16

week-old Tnip1+/+ and Tnip1-/- mice. (F) GFR of 20 week-old Tnip1-/- mice. GFR of

Tnip1+/+ littermates (n=7) served as control (100%). Data represent mean ± s.d.; *P <

0.05, **P < 0.01, ***P < 0.001; One-way Anova with Tukey’s multiple comparison test

(A); Mann-Whitney U test (C). Symbols represent data from individual mice.

0 1 0 2 0 3 0

0

2 0

4 0

6 0

8 0

1 0 0T n ip 1

- / -M y D 8 8

- / -

T n ip 1- / -

M y D 8 8+ /+

0 2 0 4 0 6 0

0

2 0

4 0

6 0

8 0

1 0 0

T n ip 1- /-

T n ip 1- /-

T L R 7- /-

T L R 9- /-

T n ip 1- /-

T L R 7- /-

T n ip 1- /-

T L R 9- /-

***ns

0 .0 0

0 .0 1

0 .0 2

0 .0 3

Sple

en/

body

weig

ht

(ratio

)

Myd88

+/+ +/+ -/- -/- -/- -/-Tnip1 +/+ -/- -/- -/- -/- -/-

Tlr-7 -9 -7/9

***

**

******

A B

D

0 2 0 4 0 6 0

0

2 0

4 0

6 0

8 0

1 0 0

***

nsns

Time

(weeks)

Surv

ival (%

) Tnip1-/-

Tnip1-/- C3-/-

Tnip1-/- Rag1-/-

0 .0 0 0

0 .0 0 5

0 .0 1 0

0 .0 1 5

0 .0 2 0

Sple

en/

body

weig

ht

(ratio

)

***

Tnip1+/+ -/- -/- -/- -/-

+/+ +/+ -/- +/+ -/-Rag1C3 +/+ +/+ +/+ -/- -/-

C

Figure S2. Survival and splenomegaly of Tnip1-/- mice with genetic deletion of TLR

proteins, Rag1 and C3

(A) Survival of mice with indicated genotype during time (Tnip1-/- MyD88+/+, n=14; Tnip1-/-

MyD88-/-, n=18; Tnip1-/- TLR9-/-, n=8, Tnip1-/- TLR7-/-, n=7; Tnip1-/- TLR9-/- TLR7-/-, n=23).

(B) Spleen/ body weight ratio of 16 week-old mice with indicated genotype. (C) Survival

of mice with indicated genotype during time (Tnip1-/-, n=22; Tnip1-/- C3-/-, n=12; Tnip1-/-

Rag1-/-, n=12). (D) Spleen/ body weight ratio of 12 week-old mice with indicated

genotype. Data represent mean ± s.d. (B, D); *P < 0.05, **P < 0.01, ***P < 0.001; ****P <

0.0001. Mantel-Cox test (A, C); Anova with Tukey’s multiple comparison test (B, D);

Symbols represent data from individual mice.

0 2 0 4 0 6 0

0

2 0

4 0

6 0

8 0

1 0 0L e g e n d

L e g e n d

L e g e n d

Time

(weeks)

Surv

ival (%

)

Time

(weeks)S

urv

ival (%

)

CD11b+ Ly6G+

(Neutro)

CD11b+ CD43low F4/80high

(Mf/ DC)

CD11b+ CD43+ F4/80low

(PMO)

CD11b

Ly6G

F4/80

CD

43

CD11c

MH

CII

0.45%

26.0%

31.2%

16.0%

47.8%

1.32%

5.75%

10.4%

82.5%

11.5% 87.7%

6.76%

10.9% 45.5%

0.6%

a

bc

a b c

94.0%

6.0%

0.0%

0.0%

6.9%

93.1%

0.0%

0.0%

Ly6C

Neutro

Ly6Chigh

DC

PMo

CD43

30.4%

36.8% 0.2%

Mf

PMo

PMo DC

0.0% 14.1%

0.0% 85.9%

Mf

B

C

CD43 F4/80

Tnip1-/-

Tnip1+/+

A

Figure S3. Characterization of immune cell populations in kidneys of Tnip1-/- mice

(A) Kidney histology (immuno-histochemistry) of 12-week-old Tnip1+/+ and Tnip1-/- mice. (B)

Flow cytometry and gating strategy of cells isolated from kidneys of Tnip1-/- mice. (C) May-

Grünwald-stained cells that were isolated by flow cytometry-based cell sorting from immune

cell populations depicted in Figure 4B. Scale bars 50 µm (A) and 20 µm (C).

0 .0

0 .5

1 .0

1 .5

2 .0 ****

0 .0

0 .2

0 .4

0 .6

0 .0

0 .1

0 .2

0 .3

0 .0

0 .1

0 .2

0 .3

0 .4* **** ****

Nr4a1 Cebpb Fcgr1 (CD64) Mafb

mR

NA

(re

l. e

xpre

ssio

n)

0 .0 0 0 0

0 .0 0 0 5

0 .0 0 1 0

0 .0 0 1 5

0 .0 0 0

0 .0 0 2

0 .0 0 4

0 .0 0 6

0 .0 0 8

0 .0 1 0

0 .0 0 0 0

0 .0 0 0 1

0 .0 0 0 2

0 .0 0 0 3

0 .0 0 0 4

0 .0 0

0 .0 5

0 .1 0

0 .1 5 ** ****

Runx1 Flt3 Ccr7 Kit

0 .0

0 .5

1 .0

1 .5

Csfr1 (CD115)

*

mR

NA

(re

l. e

xpre

ssio

n)CB

Figure S4. Characterization of PMo from kidneys, PB and BM chimeric mice

(A) Total numbers of indicated immune cell types isolated from kidneys of BM-chimeric

Tnip1+/+ recipient mice reconstituted with BM from Tnip1+/+ (WT) or Tnip1-/- (KO) mice (12

weeks after transfer).(B, C) Q-PCR-analysis of FACS-sorted Tnip1-/- tissue macrophages

(F4/80+), PMo (CD43+) and neutrophils (Ly6G+) as defined in Figure S3B. Kidneys were

obtained from chimeric mice based on BM from Tnip1-/- mice (25 weeks after BM transfer).

(D) Flow cytometry-based gating strategy of PMo from the PB of Tnip1+/+ and Tnip1-/- mice.

(E) Total numbers of indicated immune cell types isolated from the PB of BM chimeric mice

described in (A). (F) Flow cytometry analysis of cells isolated from total kidneys (left) and

purified glomeruli (right) of 25 week-old Tnip1-/- mice. Symbols represent data from

individual mice. Data represent mean ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001; ****P <

0.0001; unpaired t test (B-C); Mann-Whitney U test (A, E).

D

CD11b

Ly6G

Tnip1+/+

Tnip1-/-

PMo

Ly6Chigh

CD11b

CD

115

CD43

Ly6C

Ly6G+

8.45%

11.8%

10.7%

35.2%

29.3%

85.2%

59.5%

15.8%

18.7%

67%

A**

Neutr

ophils

(X

10

4)

PM

o(X

10

4)

0 .0

0 .5

1 .0

1 .5

0 .0

0 .2

0 .4

0 .6

0 .8

0 .0 0

0 .0 5

0 .1 0

0 .1 5

F4

/80

hig

h (X

10

4)

ns

**

Neutr

ophils

(X

10

6/m

l)

PM

o(X

10

6/m

l)

0 .0

0 .5

1 .0

1 .5

0 .0

0 .5

1 .0

1 .5

0 .0

0 .5

1 .0

1 .5

2 .0

Ly6C

hig

h (X

10

6/m

l)ns

ns

E

**

F4

/80

CD43

Total kidney Glomeruli

29%70%

9%91%

F

Figure S5. Image analysis of patrolling monocytes in human lupus nephritis

Photo montage of glomeruli from a control patient with a reperfusion renal transplant

biopsy (left side) and a patient with lupus nephritis (Class IV-G, right side). CD16 (A);

CD14 (B); CD15 (C); Periodic-acid Schiff reaction (PAS)(D); Composite of all stains with

CD16+ CD14-/dim CD15- cells indicated with white arrow heads and CD15+ cells indicated

with magenta arrowheads (E). Original magnification 400x.

Healthy control kidney Lupus nephritis

A B A B

C D DC

E E

CD16 CD16CD14 CD14

CD15 CD15PAS PAS

Composite Composite

Neutrophils

Figure S6. Cell type-specific deletion of ABIN1 in Tnip1f/f LysMcre mice and time course

of Hoxb8-FL-derived monocyte differentiation in the PB of adoptively transferred mice

(A) Immuno-blot analysis of ABIN1 expression of indicated immune cell types from 12 week-

old mice that were isolated based on flow cytometry-activated cell sorting (FACS) from BM

(neutrophils) and spleen (T-cells, B-cells), or that were in vitro generated using BM and L-cell-

conditioned medium (BMM) or GM-CSF (DC). Cells were sorted as CD11b+ Ly6G+

neutrophils, CD11b- CD3+ T-cells and CD11b- CD19+ B-cells. (B) Liver pathology of 16 week-

old mice assessed by H&E-staining. Representative images are shown. (C,D) Anemia and

splenomegaly in Tnip1fl/f LysMcre mice assessed by analysis of spleen and body weight of 12-

16 week-old mice (depicted as ratio of spleen/ body weight)(C) and analysis of hemoglobin

concentration in the peripheral blood (E). Survival of Tnip1f/f and Tnip1f/f LysMcre mice during

time. (F) Flow cytometry analysis of PB from lethally irradiated mice that were adoptively

transferred with non-differentiated Hoxb8-FL cells. PB was analyzed 5, 7 and 14 days after

Hoxb8-FL cell transfer. The upper panel shows the gating strategy for monocytes, the lower

panel shows the time course of Ly6Chigh (classical) and PMo (Ly6Clow non-classical)

monocytes in the PB.

+/+ -/- Tnip1f/f

LysMcre

DC

ABIN1

GRB2

BMM

ABIN1

GRB2

ABIN1

GRB2

ABIN1

GRB2

T cells

B cells

ABIN1

GRB2

Tnip1

A Tnip1+/+ Tnip1-/- Tnip1f/f LysMcre-/- Tnip1f/f LysMcre+/+

Liver

B

0

5

1 0

1 5

0 .0 0 0

0 .0 0 5

0 .0 1 0

0 .0 1 5

0 .0 2 0

***

****

Tnip1 +/+ -/- f/f f/f

LysMcre - - - +

Tnip1 +/+ -/- f/f f/f

LysMcre - - - +

Hb

(g/d

l)

Sp

lee

n/

bo

dyw

eig

ht ra

tio

C D

0 1 6 3 2 4 8 6 4 8 0 9 6

0

5 0

1 0 0

Su

rviv

al (%

)

Time (weeks)

Tnip1f/f

Tnip1f/f LysMcre

E

Neutro Mono

CD11b CD11b

Ly6G

CD

115

SS

C

CD

45.1

FSC FSC

Hoxb8-FL

CD43

Ly6C

d5 d7 d14

Ly6Chigh

PMo

days after transferF

0

2 0

4 0

6 0

8 0

0

2 0

4 0

6 0

8 0

B220

+ (%

)to

tal cells

(%

)

***

*** ****

Tnip1+/+ Tnip1-/-Tnip1-/- Tnip1-/-

MyD88-/-

Tnip1+/+

+

Tnip1-/-

Tnip1-/- MyD88-/-

+

Tnip1-/-

0

2 0

4 0

6 0

8 0

Ly6

Ch

igh

(%

)

*****

Tnip1+/+ Tnip1-/-Tnip1-/- Tnip1-/-

MyD88-/-

Tnip1+/+

+

Tnip1-/-

Tnip1-/- MyD88-/-

+

Tnip1-/-

Tnip1+/+ Tnip1-/-Tnip1-/- Tnip1-/-

MyD88-/-

Tnip1+/+

+

Tnip1-/-

Tnip1-/- MyD88-/-

+

Tnip1-/-

0

2 0

4 0

6 0

8 0

CD

3+

(%

)

**

Tnip1+/+ Tnip1-/-Tnip1-/- Tnip1-/-

MyD88-/-

Tnip1+/+

+

Tnip1-/-

Tnip1-/- MyD88-/-

+

Tnip1-/-

Total cells Ly6Chigh Mono

B-cells T-cells

Figure S7. ABIN1 controls cell-intrinsically MyD88-mediated PMo deregulation

Cell numbers of total white blood cells (WBC), Ly6Chigh monocytes, B220+ B-cells and

CD3+ T-cells in the PB blood of BM chimeras (6 weeks after transfer) which were

established with BM from Tnip1+/+ and Tnip1-/- mice, or from BM from Tnip1-/- and

Tnip1-/- Myd88-/- mice. Relative cell numbers derived from Tnip1+/+ vs. Tnip1-/- BM and

Tnip1-/- vs.Tnip1-/- Myd88-/- BM are shown.

Data represent mean ± s.d.; *P < 0.05, **P < 0.01, Mann-Whitney U test. Symbols

represent data from individual mice.

BM: BM:

BM: BM:

Tnip1-/-

Nr4a1-/-

Figure S8. Genetic suppression of PMo protects from GN

(A) Flow cytometry analysis of PB from 12-week-old mice with indicated genotype.

CD11b+ Ly6G- CD115+ monocytes are shown. (B-E) Kidney pathology and function of

16-week-old Tnip1-/- and Tnip1-/- Nr4a1-/- mice as analyzed by histology based on H&E-

staining and antibodies against C3 and IgG (B), CD43 (C), the measurement of avg.

glomerulus size (D) and protein concentration in urine (E). Representative glomeruli are

shown. (F) Total numbers of CD43+ PMo, F4/80high myeloid cells and Ly6G+ neutrophils

isolated from the kidneys of 16-old Tnip1-/- and Tnip1-/- Nr4a1-/- mice. (G) Serum levels

of anti-dsDNA Ig and ANA of 16-week-old Tnip1-/- and Tnip1-/- Nr4a1-/- mice. (H,I)

Anemia and splenomegaly in Tnip1-/- and Tnip1-/- Nr4a1-/- mice assessed by analysis of

spleen and body weight of 16 week-old mice (depicted as ratio of spleen/ body

weight)(H) and analysis of hemoglobin concentration in the peripheral blood (G). (J)

Survival of Tnip1-/- and Tnip1-/- Nr4a1-/- mice during time. Scale bars 20 µm. Data

represent mean ± s.d.; *P < 0.05, **P < 0.01; One-way Anova with Tukey’s multiple

comparison test (D-I). Symbols represent data from individual mice. Ns=not significant.

Tnip1-/-

Nr4a1+/+

C3

IgG

H&E

B

D

FG

lom

eru

lus a

rea (

µm

2)

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

*** **

Neutr

o(X

10

4)

F40/8

0h

igh

(X10

4)

PM

o(X

10

4)

0 .0

0 .5

1 .0

1 .5

2 .0

0 .0

0 .1

0 .2

0 .3

0 .0

0 .5

1 .0

1 .5

2 .0

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

*** **ns ns**** ****

E

G

0

1

2

3

4

5

Pro

tein

uria S

core

(0-4

)

**** ****

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

Anti-d

sD

NA

Ig (

U/m

l)

0

5 0

1 0 0

1 5 0

2 0 0

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

*** ns

AN

A Ig

(U/m

l)

0

5 0

1 0 0

1 5 0

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

** ns

C

Tnip-/-

Nr4a1-/-

Tnip-/-

Nr4a1+/+

CD43

Ly6

C

CD43

PMo

Tnip1+/+

Nr4a1+/+

Tnip1+/+

Nr4a1 -/-Tnip1-/-

Nr4a1 +/+

Tnip1-/-

Nr4a1 -/-

56.6%

25.6%

74.8%

5.06%

26.6%

53.3%

74.4%

2.95%

Ly6Chigh

A

0 .0 0

0 .0 1

0 .0 2

0 .0 3

0 .0 4

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

Sple

en/

Bodyw

eig

ht

ratio

H

0 5 1 0 1 5 2 0

0

2 0

4 0

6 0

8 0

1 0 0

0 5 10 15 200

20

40

60

80

100 Tnip1-/-

Tnip1-/-

Nur77-/-Tnip1-/- Nr4a1-/-

Tnip1-/-

Time (weeks)

Surv

ival (%

)

J***ns

0

5

1 0

1 5

I

Tnip1

Nr4a1

+/+ +/+ -/- -/-

+/+ -/- +/+ -/-

Hb

(g/d

l)

*ns