pearson r = 0.81 1 0 0 2 µm 2 0 0 0 · 2019-05-21 · 0 = normal 1 = minimal 2 = mild 3 = moderate...
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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
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