Embed Size (px)
Citation preview
0 1 2 3 4 5 6 775
85
95
105
3
4
5
6
7
8
9
0 1 2 3 4 5 6 70.0
0.5
1.0
1.5
Wei
ght (
% S
tarti
ng)
Sco
re
Lyn+/+
Lyn-/-
Time (days)
A B
0 1 2 3 4 5 6 7 880
85
90
95
100
105
Time (days)
Leng
th (c
m)
*
Lyn+/+
Lyn-/-
Untreated DSS
C Weight Change Rectal Bleeding Colon Length
Lyn+/+
Lyn-/-
D
200 m
DSS 4x 10x
Lyn-/-
Untreated 4x 10x
Lyn+/+
200 m 500 m
500 m
200 m 500 m
500 m 200 m
0 1 2 3 4 5 6 7 880
85
90
95
100
105
* ** *** *** *
Supplemental Figure 1
*** ***
Figure S1. Lyn-deficiency increases susceptibility to DSS-induced colitis. Lyn +/+ and Lyn -/- mice were challenged with 2.5% DSS and (A) body weight and (B) rectal bleeding were monitored for 7 days. Mice were sacrificed on day 7, (C) colon length was measured and (D) cross sections of distal colon were stained with hematoxylin and eosin. Representative data from three independent experiments are shown, n=4-9, error bars represent SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.005 . No differences in weights of untreated Lyn+/+ and Lyn-/- mice were observed (data not shown).
0 102 103 104 105
<PE-A>: CD4
0
102
103
104
105
<PE-
Cy7
-A>:
CD
8
0 102 103 104 105
<PE-A>: CD4
0
102
103
104
105
<PE-
Cy7
-A>:
CD
8
0 102 103 104 105
<FITC-A>: CD3
0
102
103
104
105
<Pac
ific
Blu
e-A
>: C
D45
0 102 103 104 105
<FITC-A>: CD3
0
102
103
104
105
<Pac
ific
Blu
e-A
>: C
D45
0 102 103 104 105
<PE-A>: CD4Bt
0
102
103
104
105
<PE-
Cy7
-A>:
CD
8
H20
DSS
CD45+ CD45+CD11c+MHCII+ CD45+CD11c+MHCII+
Lyn+/+ Lynup/up A
CD45+
CD11b
CD
103
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CD11c
MH
C II
20.8 ± 2.4 30.2 ± 4.0
15.4 ± 1.6 25.0 ± 2.0
H20
DSS
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CD11b
Gr-
1
0.2 ± 0.1
2.6 ± 0.7
0.6 ± 0.1
4.1 ± 0.2
7.4 ± 0.2 7.6 ± 1.2
10.7 ± 0.8 10.5 ± 2.4
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CD11b
F4/8
0
32.5 ± 1.7 37.8 ± 3.1
16.0 ± 6.0 23.8 ± 7.2
0 102 103 104 105
<FITC-A>: CD3
0
102
103
104
105
<Pac
ific
Blu
e-A
>: C
D45
0 102 103 104 105
<PE-A>: CD4Bt
0
102
103
104
105
<PE-
Cy7
-A>:
CD
8
0 102 103 104 105
<FITC-A>: CD3
0
102
103
104
105
<Pac
ific
Blu
e-A
>: C
D45
CD45+CD3+
CD3
CD
45
CD45+
CD4
CD
8 53.0 ± 4.6 54.2 ± 11.9
16.1 ± 2.2 35.2 ± 3.0
17.5 ± 3.3 29.4 ± 4.5
21.9 ± 1.0 24.3 ± 2.5
56.0 ± 3.6 62.3 ± 4.4
22.5 ± 5.7 26.5± 6.7
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
36.1 ± 5.9 38.9 ± 7.3
44.9 ± 8.6 45.7 ± 10.0
26.8 ± 3.8 26.5 ± 1.4
44.8 ± 4.4 50.9 ± 5.0
7.8 ± 0.9 10.2 ± 1.3
4.7 ± 0.3 6.8 ± 0.3
0 102 103 104 105
<APC-A>: B220
0
102
103
104
105
<FIT
C-A
>: C
D19
0 102 103 104 105
<APC-A>: B220
0
102
103
104
105
<FIT
C-A
>: C
D19
0 102 103 104 105
<APC-A>: B220
0
102
103
104
105
<FIT
C-A
>: C
D19
0 102 103 104 105
<APC-A>: B220
0
102
103
104
105
<FIT
C-A
>: C
D19
B220
CD
19
13.4 ± 2.7 1.0 ± 0.8
12.6 ± 1.9 0.7 ± 0.3
CD45+
H20
DSS
0 102 103 104 105
<PE-A>: CD80
0
20
40
60
80
100
% o
f Max
0 102 103 104 105
<FITC-A>: CD86
0
20
40
60
80
100
% o
f Max
CD80 CD86
Lyn+/+
Lynup/up
0 102 103 104 105
<PE-A>: CD80
0
20
40
60
80
100
% o
f Max
0 102 103 104 105
<FITC-A>: CD86
0
20
40
60
80
100
% o
f Max
CD45+
010203050
200350500650800950
B
Num
ber (
x103
)
Total Numbers
Lyn+/+
Lynup/up
Supplemental Figure 2
Lyn+/+ Lynup/up
Lyn+/+ Lynup/up
Figure S2. The intestinal immune compartment of Lyn up/up mice. Lyn +/+ and Lyn up/up mice were left untreated (H2O) or were challenged with 2.5% DSS for 7 days (DSS). Colonic lamina propria cells were isolated and analyzed for the presence of macrophages (CD11b+F4/80+), granulocytes (Gr-1+CD11b+), DCs (CD11c+MHCII+, CD103+/CD11b+, CD80 and CD86), T cells (CD3+, CD4+/CD8+), and B cells (CD19+B220+) by flow cytometry. For CD80 and CD86 expression, shaded histograms represent CD11c-MHCII- cells. Lyn +/+ (grey) and Lyn up/up (black) DCs (CD11c+MHCII+) are represented by open histograms. All cells were first gated on live (propidium iodide negative) CD45+ populations. (A) Representative plots from 3 independent experiments are shown. Numbers represent pooled mean frequency ± SEM, n=6. (B) Graphs indicate total numbers of indicated cell populations per colon. Data is pooled from from 3 experiments, n=6, and error bars represent SEM.
CD45+ CD45+ CD45+ Rag-/- Rag-/-Lynup/up
H20
DSS
CD11c
MH
C II
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
64.3 ± 2.3 61.6 ± 3.6
64.0 ± 0.7 58.7 ± 0.9
CD11b
Gr-
1
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
7.5 ± 0.8
0.6 ± 0.2
10.0 ± 1.1
1.5 ± 0.1
22.7 ± 0.9
5.2 ± 1.5
14.5 ± 1.7
8.7 ± 2.8
CD11b
F4/8
0
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
10552.0 ± 2.6 39.3 ± 7.8
55.6 ± 3.5 60.2 ± 2.3
Supplemental Figure 3
Rag-/- Rag-/-Lynup/up Rag-/- Rag-/-Lynup/up
Figure S3. The intestinal immune compartment of Rag-/-Lyn up/up mice. Rag-/- and Rag-/-Lyn up/up mice were left untreated (H2O) or were challenged with 2.5% DSS for 7 days (DSS). Colonic lamina propria cells were isolated and analyzed for the presence of macrophages (CD11b+F4/80+), granulocytes (Gr-1+CD11b+), and DCs (CD11c+MHC II+). All cells were first gated on live (propidium iodide negative) CD45+ populations. Representative plots from 2-3 independent experiments are shown. Numbers represent pooled mean frequency ± SEM, n=4-6.
Supplemental Figure 4
Unstim.
IL-23 + LPS
CD45+IL-22+Lin-CD90+ CD45+
IL-22
CD
90
CD45+IL-22+CD90+
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
MFI 493
0 102 103 104 105
<PE-A>: IL-22
0
102
103
104
105
<APC
-A>:
CD
90
MFI 620 59.2 ± 5.3 72.1 ± 6.6
67.8 ± 7.9 69.1 ± 11.1
0 102 103 104 105
0
102
103
104
105
MFI 1204
0 102 103 104 105
<PE-A>: IL-22
0
102
103
104
105
<APC
-A>:
CD
90
MFI 1713 85.3 ± 1.3 89.2 ± 1.8
69.5 ± 1.6 68.7 ± 1.2
CD90
Line
age
CD25
CD
4
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
MFI 1006
MFI 1349 82.4 ± 1.9 86.9 ± 1.7
70.5 ± 1.5 70.0 ± 2.0
IL-23
Rag-/- Rag-/-Lynup/up Rag-/- Rag-/-Lynup/up Rag-/- Rag-/-Lynup/up
Figure S4. Lyn regulates IL-22 production from ILCs, the major source of IL-22 in splenic cultures. Splenocytes from naïve Rag-/- and Rag-/-Lyn up/up mice were treated with brefeldin A alone (Unstim.) or brefeldin A and IL-23 ± LPS for 8 hours and IL-22 production was assessed by flow cytometry. Plots represent data pooled from 2 independent experiments, n=4. Numbers on plots represent pooled mean frequency ± SEM or representative mean fluorescence intensities (MFI).
Supplemental Figure 5
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
<APC-A>: CD11c
0
102
103
104
105
<PE-
A>: C
L2
0 102 103 104 105
<APC-A>: CD11c
0
102
103
104
105
<PE-
A>: C
L2
0 102 103 104 105
<APC-A>: CD11c
0
102
103
104
105
<PE-
A>: C
L2
0 102 103 104 105
<APC-A>: CD11c
0
102
103
104
105
<PE-
A>: C
L2
33.8 1.7 11.0 19.1
1.5 0.1 0.4 0.5
CD11c
MH
C II
PBS 2 Days 3 Days 4 Days
Spleen
cLP
B
C
0 102 103 104 105
<APC-A>: CD90
0
102
103
104
105
<PE
-A>:
Lin
eage
0 102 103 104 105
<APC-A>: CD90
0
102
103
104
105
<PE
-A>:
Lin
eage
0 102 103 104 105
<PE-Cy7-A>: CD11c
0
102
103
104
105
<APC
-Cy7
-A>:
MH
C II
0 102 103 104 105
<PE-Cy7-A>: CD11c
0
102
103
104
105
<APC
-Cy7
-A>:
MH
C II
0 102 103 104 105
<FITC-A>: CD11c-GFP
0
102
103
104
105
<APC
-Cy7
-A>:
MH
C II
0 102 103 104 105
<FITC-A>: CD11c-GFP
0
102
103
104
105
<APC
-Cy7
-A>:
MH
C II
0 102 103 104 105
<APC-A>: CD90
0
102
103
104
105
<PE
-A>:
Lie
nage
0 102 103 104 105
<APC-A>: CD90
0
102
103
104
105
<PE
-A>:
Lie
nage
0 102 103 104 105
<PE-Cy7-A>: CD11c
0
102
103
104
105
<APC
-Cy7
-A>:
MH
C II
0 102 103 104 105
<PE-Cy7-A>: CD11c
0
102
103
104
105
<APC
-Cy7
-A>:
MH
C II
0 102 103 104 105
<APC-A>: CD90
0
102
103
104
105
<PE
-A>:
Lin
eage
0 102 103 104 105
<APC-A>: CD90
0
102
103
104
105
<PE
-A>:
Lin
eage
PBS
CD45+ DT
Spleen MLN cLP PBS DT PBS DT
CD90
Line
age
CD11c
MH
C II
1.2 0.1
0.3 0.4
1.6 0.1
0.3 1.6
8.2 1.3
2.2 2.8
CD90
Line
age
0 102 103 104 105
R1-A: CD90 - APC-A
0
102
103
104
105
B1-A
: Lin
eage
- FI
TC-A
0 102 103 104 105
R1-A: CD90 - APC-A
0
102
103
104
105
B1-A
: Lin
eage
- FI
TC-A
12.8 0.0 0 102 103 104 105
R1-A: CD90 - APC-A
0
102
103
104
105
B1-A
: Lin
eage
- FI
TC-A
0 102 103 104 105
R1-A: CD90 - APC-A
0
102
103
104
105
B1-A
: Lin
eage
- FI
TC-A
25.5 0.0 0 102 103 104 105
R1-A: CD90 - APC-A
0
102
103
104
105
B1-A
: Lin
eage
- FI
TC-A
0 102 103 104 105
R1-A: CD90 - APC-A
0
102
103
104
105
B1-A
: Lin
eage
- FI
TC-A
22.2 6.8
A PBS
CD45+ anti-CD90
Spleen MLN cLP
PBS anti-CD90 PBS anti-CD90
Figure S5. Systemic administration of monoclonal anti-CD90 antibodies or DT depletes CD90+ cells and DCs, respectively. (A) Rag -/- and Rag-/-Lyn up/up mice were mock injected (PBS) or treated with anti-CD90 mAb. Two days later mice were injected with LPS and presence of CD90+ cells in the spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) was assessed by flow cytometry 2 hours post-LPS injection. (B) Cd11cDTR mice were injected with PBS or diptheria toxin (DT) and the presence of DCs (CD11c+MHC II+) in the cLP and spleen was assessed by flow cytometry at the indicated time points post-DT injection. (C) Cd11cDTR and Cd11cDTR-Lynup/up mice were mock injected (PBS) or treated with DT 1 day prior to LPS injection and presence of DCs (CD11c+MHC II+) and ILCs (lineage-CD90+) cells in the spleen, MLN and cLP was assessed by flow cytometry. All cells were first gated on live (propidium iodide negative) CD45+ populations. Representative plots are shown and numbers represent frequency of highlighted population.
100 101 102 103 104
PE-A: Lyn PE-A
0
20
40
60
80
100%
of M
ax
100 101 102 103 104
APC-A: Lyn APC-A
0
20
40
60
80
100
% o
f Max
100 101 102 103 104
PE-A: Lyn PE-A
0
20
40
60
80
100
% o
f Max
100 101 102 103 104
PE-A: Lyn PE-A
0
20
40
60
80
100
% o
f Max
T Cells (TCR +)
Macrophages (CD11b+F4/80hi)
Neutrophils (CD11b+Gr-1hi)
NK Cells (NK1.1+DX5+)
Lyn+/+
Lyn-/-
Lyn 100 101 102 103 104
PE-A: Lyn PE-A
0
20
40
60
80
100
% o
f Max
(CD45-)
Supplemental Figure 6
Figure S6. Colonic expression of Lyn. Colonic lamina propria cells were isolated from naïve Lyn +/+ (black open histograms) and Lyn -/- (grey filled histograms) mice and Lyn expression in conventional T cells (TCR +), macrophages (CD11b+F4/80hi), neutrophils (CD11b+Gr-1hi), NK cells (NK1.1+DX5+) and non-hematopoietic cells (CD45-) was assessed by flow cytometry. All cells were first gated on live CD45+ populations, with the exception of CD45- cells which were gated on live CD45- cells. Representative histograms are shown.
