supplemental material supplemental figure 1. further analysis of … · 2013-04-17 · ! 1!...
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Supplemental Material
Supplemental Figure 1. Further Analysis of CX3CR1 Expressing Myeloid Cells in
Resting CX3CR1gfp/+ Mice
(a) The mean frequency of CX3CR1-, CX3CR1int and CX3CR1hi CD11b+ cells amongst the
live-gated CD45+ fraction of colonic LP cells. (b) FSC and SSC profile of live-gated CD45+
CX3CR1-, CX3CR1int and CX3CR1hi CD11b+ cells. (c) SSC profile and expression of
SiglecF by CX3CR1- CD11b+ LP cells. (d) Expression of CD64 by P1 to P5 subsets of
colonic myeloid cells. (e) CX3CR1 expression by resident colonic LP mφ (P4), M-CSF
generated BM-derived mφ and resident peritoneal mφ. (f) Expression of CX3CR1 by P1 to
P5 subsets of colonic myeloid cells (see text for details), compared with
Ly6CloCX3CR1+CCR2- and Ly6ChiCX3CR1intCCR2+ blood monocytes. (g) The mean
frequency of P1, P2, P3 and P5 subsets as a proportion of the total CX3CR1int compartment
in colon.
Supplemental Figure 2. Effects of Flt3L Administration on Colonic Myeloid Cells
(a) CX3CR1+/gfp mice received flt3L for 8 consecutive days and the absolute numbers of cells
in populations 1-5 of CX3CR1 expressing colonic LPL compared with those in untreated
CX3CR1+/gfp mice. Only the F4/80- CD11c+ subset of CX3CR1int cells (P5) shows significant
expansion, (b) Expansion of CD103+ MHCII+ DC in colon after treatment with flt3L. Data
are representative of two individual experiments with 3 mice per group. (*** p<0.0001)
Supplemental Figure 3. Uptake of BrdU by Colonic Myeloid Cells
CX3CR1+/gfp mice received 1mg BrdU ip and 3, 12, 24 and 48h later, BrdU uptake was
assessed in colonic CX3CR1int (P1-P3) and CX3CR1hi (P4) populations. Representative
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dotplots of BrdU and CD11b staining in gated cells from BrdU injected mice and in non-
injected controls.
Supplemental Figure 4. Alternative Gating Strategy for Identifying Colonic Myeloid
Cell Subsets in Non-CX3CR1+/gfp Mice
(a) CD11b+ cells were selected from the CD45+7-AAD- fraction of colonic digests from WT
mice and granulocytes gated out using forward (FSC) and side scatter (SSC) profiles. Ly6C
and MHCII expression identified four populations (A-D). (b) Applying the same gating to
CX3CR1gfp/+ mice shows that population A (CD11b+Ly6ChiMHCII-) is equivalent to P1
identified previously in CX3CR1+/gfp mice and population B (CD11b+Ly6C+MHCII+) is
identical to P2. Population C in WT mice is the F4/80+ fraction of the MHCII+ CD11b+SSClo
cells and contains both the previously identified P3 and P4 subsets in CX3CR1+/gfp.
Population D in WT mice is the CD11c+ fraction of the MHCII+ CD11b+SSClo cells and
contains all CD11b+ DC (CX3CR1neg and CX3CR1int) and not only the CX3CR1int DC
designated P5 in CX3CR1+/gfp mice.
Supplemental Figure 5: Purification and Adoptive Transfer of BM Monocytes
(a) 'Inflammatory' Ly6Chi (Ly6ChiCX3CR1int) and 'resident' Ly6Clo (Ly6CloCX3CR1+)
monocytes were identified amongst the CD11b+Ly6G-CD117- fraction of BM cells sorted by
FACS. (b) Presence of donor derived cells in colon and bloodstream of CCR2-/- mice 96hr
after transfer of 1x106 purified Ly6Clow BM monocytes from CD45.1+/CD45.2+ CX3CR1+/gfp
mice. (c) Migration of donor monocytes into resting, unmanipulated colon. 2x106 purified
Ly6Chi or 1x106 purified Ly6Clo BM monocytes from CD45.1+/CD45.2+ CX3CR1+/gfp mice
were transferred into resting CX3CR1+/gfp (CD45.2) mice and the presence of donor cells
assessed in colon 24 hours later. The expression of CX3CR1 on donor derived and
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endogenous CD11b+ colonic cells was assessed in wild type recipients of Ly6Chi monocytes
(right panel). (d) Presence of donor derived cells in colon and bloodstream of colitic WT
(CD45.1) mice 72hr after transfer of 1x106 purified Ly6Clo BM monocytes from
CD45.1+/CD45.2+ CX3CR1+/gfp mice on day 3 of DSS colitis.
Supplemental Figure 6. Recruitment of Ly6Chi Monocytes to Colon of CD11c-DTR
Mice
(a) Depletion of colonic mononuclear cells 24 hours after treatment of CD11c-DTR-GFP
mice with DT and in untreated controls. Representative Ly6C and MHCII expression by
CD11b+SSClo leucocytes (top panels); discrimination of mφ and DC amongst Ly6C- MHCII+
cells by expression of F4/80 and CD11c (bottom panels). (b) Donor-derived cells
(CD45.1+/CD45.2+ CX3CR1+/gfp) amongst live-gated CD45+ CD11b+ colonic LP cells 24 and
96h after transfer of 2x106 FACS sorted Ly6Chi BM monocytes into CD11c-DTR-GFP mice
given DT 24h before transfer. Note the presence of endogenous CD11c-GFPloCD45.1- cells
in CD11c-GFP mice. (c) Expression of Ly6C, MHCII and F4/80 on transferred donor
monocytes (upper panels) and on donor-derived cells isolated from the recipient colon (lower
panel), 24h (narrow line) or 96h (broad line) after transfer. Shaded histograms represent
isotype staining. (d) Expression of CX3CR1-GFP by donor-derived cells isolated from the
recipient colon at 24h (narrow line) or 96h (broad line) after transfer, compared with total
live-gated CD45+ CD11b+ LP leucocytes from a resting CX3CR1+/gfp mouse (shaded
histogram). Forward scatter of donor cells at each time point (right panel). (e) Presence of
donor Ly6Chi monocytes (CD45.1+/CD45.2+ CX3CR1+/gfp) in bloodstream of CD11c-DTR-
GFP mice 24h and 96h after transfer.
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Supplemental Figure 7. Cytokine Production by Colonic CX3CR1-defined Myeloid
Populations in Healthy and Inflamed Intestine
(a) Colonic LP cells isolated from resting CX3CR1+/gfp mice, or from mice receiving 2% DSS
for 4 days, were cultured for 4.5h in medium before IL10 and TNFα production were
assessed by intracellular cytokine staining. Results shown are representative IL10 and TNFα
expression by each CX3CR1-defined population in resting (top panel) or inflamed colon
(lower panel)). (b) Representative IL10-eGFP expression and the mean percentage of IL10-
eGFP+ cells within the indicated populations of colonic myeloid cells obtained from resting
Vert-X mice. Results are means of 3-4 mice and are representative of three independent
experiments.
Supplemental Figure 8. Gene Expression by Colonic CX3CR1+ Subsets During Colitis
Quantitation of mRNA for different genes in P2 (a), P3 (b) and P4 (c) subsets of colonic
myeloid cells sorted from CX3CR1gfp/+ mice on day 4 of colitis assessed by qRT-PCR.
Results shown are mean expression relative to cyclophilin A using the 2-ΔC(t) method. The
mean was obtained from three independent experiments using cells pooled from eleven mice.
Supplemental Figure 9. Expression of TLR by CX3CR1+ Myeloid Subsets in Steady
State Colon
(a) qRT-PCR analysis of the expression of TLR1-9 by FACS-purified colonic populations 1
to 4 from CX3CR1gfp/+ mice compared with M-CSF generated BM mφ (BMM). Results
shown are the mean expression relative to cyclophilin A (CPA). The mean was obtained
from three independent experiments using cells pooled from 11-12 mice per experiment. (b)
Expression of TLR2 (upper panel) and CD14 (lower panel) by P1-P4 isolated from resting
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CX3CR1+/gfp mice assessed by flow cytometry. Shaded histograms represent staining with
the appropriate isotype controls.
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Supplemental Table 1. Clinical details of Crohn’s disease patients used
Pat Age (Yrs) Sex
Duration of Disease (Years) Involvement Smoker
CRP (mg/L)
Fecal calprotectin (mg/kg) Therapy
1 23 F 4 Ileo-colonic No 19 1002 -
2 25 M 13 Jejunal, Ileal,
Colonic No 22 571 Mtx, Bud
3 38 M 2 Ileo-cecal No 74 956 -
4 31 M 9 Ileal No 42 828 -
5 32 M 2 Ileal Yes 16 133 TNF
6 34 M 22 Ileo-colonic No n.a. n.a. -
7 27 F 6 Ileo-colonic No 33 782 Aza
Aza, Azathioprine; Bud, Budesonide; CRP, C-reactive protein; n.a., not available; Mtx, Methotrexate; TNF, Tumour necrosis factor α inhibitor
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Supplementary Table 2. List of Antibodies
Antibody Clone Source
Mouse
CD11c HL3 BD Biosciences CD14 Sa2-8 eBioscience
CD16/32 2.4G2 BD Biosciences CD45 30-F11 BD Biosciences CD45.1 A20 BD Biosciences CD45.2 104 BD Biosciences
CD103 M290 BD Biosciences CD103 2E7 eBioscience CD115 AFS98 eBioscience
CD117 2B8 BD Biosciences CD206 MR5D3 Biolegend BrdU BD Biosciences F4/80 BM8 eBioscience
IL-10 JES5-16E3 BD Biosciences Ly6C AL-21 BD Biosciences Ly6G 1A8 BD Biosciences
MHC II (IA-IE) M5/114.15.2 eBioscience SiglecF E50-2440 BD Biosciences TLR2 6C2 eBioscience
TNFα MP-6XT22 BD Biosciences
Human
CD3 UCHT1 eBioscience CD11c 3.9 eBioscience/Miltenyi Biotec
CD14 61D3 eBioscience CD19 HIB19 eBioscience CD20 2H7 Biolegend CD45 H130 eBioscience
CD56 Alpha Diagnostic Intl. Inc. CD103 Ber-ACT8 or
B-Ly7 eBioscience
CD163 eBGHI/61 or BerMac3
eBioscience/DAKO
CD209 eB-h209 or DCN-46
eBioscience/BD Biosciences
HLA-DR L43 eBioscience
TCRab IP26 Biolegend
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Supplemental Table 3. Primers used for qRT-PCR
Gene Sense Anti-Sense
Cyclophilin A GTG GTC TTT GGG AAG GTG AA TTA CAG GAC ATT GCG AGC AG
Cx3cr1 TGT CCA CCT CCT TCC CTG AA TCG CCC AAA TAA CAG GCC
Cd163 CCT TGG AAA CAG AGA CAG GC TCC ACA CGT CCA GAA CAG TC
Cd206 TGT GGT GAG CTG AAA GGT GA CAG GTG TGG GCT CAG GTA GT
Tgfbr2 ACA TTA CTC TGG AGA CGG TTT GC AGC GGC ATC TTC CAG AGT GA
Arginase-1 CAG AAG AAT GGA AGA GTC AG CAG ATA TGC AGG GAG TCA CC
Ccr2 ATC CAC GGC ATA CTA TCA ACA TC CAA GGC TCA CCA TCA TCG TAG
Il6 CCA GTT GCC TTC TTG GGA CT GGT CTG TTG GGA GTG GTA TCC
inos GCC ACC AAC AAT GGC AAC A GCC ACC AAC AAT GGC AAC A
Il10 GCT CTT ACT GAC TGG CAT GAG CGC AGC TCT AGG AGC ATG TG
Tnfa ACC CTC ACACTC AGA TCA TCT TC TGG TGGTTT GCT ACG ACG T
Il1b CTG GTG TGT GAC GTT CCC ATT A CCG ACA GCA CGA GGC TTT
Vegf CCT TCG TCC TCT CCT TAC CC AAG CCA CTC ACA CAC ACA GC
Tlr1 TGG ACA CCC CTA CAG AAA CGT AAT TTG GTT TAG TCA TTG TTG
TAT GGC C Tlr2 CTG GAG CAT CCG AAT TGC A CAT CCT CTG AGA TTT GAC GCT TT
Tlr3 CCA GAA GAA TCT AAT CAA ATT AGA
TTT GTC
TTT TGC TAA GAG CAG TTC TTG
GAG Tlr4 GGC AAC TTG GAC CTG AGG AG CAT GGG CTC TCG GTC CAT AG
Tlr5 CAC TCC CTC GGA GAA CCC A GGC CTT GAA AAA CAT CCC AAC
Tlr6 AAA GTC CCT CTG GGA TAG CCT CT TGC TTC CGA CTA TTA AGG CCA
Tlr7 ACA GAA ATC CCT GAG GGC ATT CAG ATG GTT CAG CCT ACG GAA G
CX3CR1neg CX3CR1int CX3CR1high0
5
10
15
20
% o
f CD
45 L
ive
cells
b c
d
FSC
SS
C
CX3CR1int CX3CR1hi CX3CR1neg
Ly6Chi blood monocytes
Ly6Clo blood monocytes
P1
P2
P3
P4
P5
SS
C
SiglecF
CX3CR1hi LP mφ [P4]
Resident Peritoneal mφ
M-CSF BM-derived mφ
e
P1 P2 P3 P5
0
10
20
30
40
% o
f CX
3CR
1int c
ells
a
f
CX3CR1-GFP
CX3CR1-GFP
!!
!!
!!
!!
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!!
!!
!!
P1
P2
P3
P4
P5
!!CD64
g
Supplementary Figure 1
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a Ly6ChiMHCneg [P1]
Ly6C+MHC+ [P2]
F4/80+ MHC+ [P3]
CX3CR1hi [P4]
CX3CR1int DC [P5]
b Gated: CD45+ 7-AADneg
63.6
33
19.2
77.7
P3
P5
Resting CX3CR1+/gfp CX3CR1+/gfp + flt3L
CD11c
F4/
80
Gated: CD45+ 7-AADneg CD11b+ CX3CR1int Ly6Cneg MHCII+
2.6 27
Resting CX3CR1+/gfp CX3CR1+/gfp + flt3L
Resting flt3L0
1
2
3
Num
ber C
X3C
R1hi
live
cel
lspe
r co
lon
(x10
5 )
Resting flt3L0
20
40
60
80
100
120
Num
ber F
4/80
+ MH
C+ Ly
6Cne
g
CX
3CR
1int c
ells
per
col
on (x
103 )
Resting flt3L0
20
40
60
80
100
120
Num
ber o
f Ly6
C+ M
HC
+liv
e ce
llspe
r col
on (x
103 )
Resting flt3L0
5
10
15
20
***
Num
ber
CD
103+
DC
per
colo
n (x
105 )
Resting flt3L0
20
40
60
80
100
120
Num
ber o
f Ly6
Chi
live
cells
per c
olon
(x10
3 )
Resting flt3L0
20
40
60
80
100
120***
Num
ber C
D11
c+ MH
C+ Ly
6Cne
g
CX
3CR
1int c
ells
per
col
on (x
103 )
MHC II
CD
103
Supplementary Figure 2
a
BrdU
CD
11b
Non-injected control 3h 12h
[P1]
24h 48h
[P2]
[P3]
[P4]
1.4 0.5 5.4 33.9 20.3
1.9 0.6 1.4 1.2 25.5
2.2 1.0 0.7 1.7 13.7
1.5 2.2 1.4 1.2 2.3
Supplementary Figure 3
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FSC
CD
11b
a
b
Gated: CD45+7-AADneg
CX3CR1-GFP
A B C
D
C = F4/80+MHCII+CD11cint/+ [P3+P4]
A = Ly6Chi monocytes [P1]
B = Ly6C+MHC+ [P2]
D = CD11b+DC [incl. P5]
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!!
FSC
SS
C
MHC II
Ly6C
CD11c
F4/
80
Supplementary Figure 4
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a
CX3CR1-GFP
Ly6C
45.6
8.7
97.0
0.1
0
97.9
Pre-sort
Post-sort
Ly6Chi Ly6Clo
Gated: CD11b+ Ly6Gneg CD117neg
c
0.12 0.001
Blood Colonic LP
0.018 0.036 0.013
PBS Ly6Clo
monocytes
CX3CR1-GFP
CD11b+ cells from resting CX3CR1+/gfp
Ly6Chi donor
Ly6Chi monocytes
Blood Colonic LP
CCR2-/- Recipients – Ly6Clo monocytes
d
b
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CD45.1+ WT Colitic Recipients – Ly6Clo monocytes
0.18
0.001
0.006
0
0.005 0
Resting CX3CR1+/gfp Recipients
CX3CR1-GFP
CD
45.1
CD45.1
CD
45.2
CX3CR1-GFP
CD
45.2
Supplementary Figure 5
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a PBS + DT b 0 0 0.4 0 0.1 0.3
d
PBS 24h 96h
Ly6C MHC II F4/80
24h
96h
c
CX3CR1-GFP
0.001 0 0.18 0 0.09 0
e PBS 24h 96h
FSC
Input phenotype
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25
42
75
14 10
CD11b+ cells resting CX3CR1+/gfp
24h 96h
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Acquired phenotype
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Supplementary Figure 6
MHC II
Ly6C
CD11c
F4/
80
GFP
CD
45.1
GFP
CD
45.1
!!
!!
!!
!!
!!
!!
!!
!!
a Ly6Chi [P1]
Ly6C+MHC+ [P2]
F4/80+ MHC+ [P3]
CX3CR1hi [P4]
!
!
!!
!
!
!
!
5.9
5.0
20.6 16.9
10.1
19.4 8.7
13.9
9.8 13.4
17.9
10.4
!!!!
!!
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9.03
4.4
38.5 28.2
7.9
32.3 29.9
15.4
21.2 12.9
21.1
7.4
IL10
TN
Fα
b 3.7 17.5 60.1 2.9
Ly6Chi [P1] Ly6C+MHC+ [P2] Ly6CnegMHC+ [P3] + CX3CR1hi [P4] CD11b+ DC
Colitic
Resting
IL10 eGFP
CD
11b
Supplementary Figure 7
P1 P2 P3/P4 DC0
20
40
60
80
100
*** ******
% IL
10-e
GFP
+ CD
11b+ c
ells
a
Cx3cr1 Cd163 Cd206 Tlr2 Tlr4 Ccr2 Il10 Il6 Tnfa0.01
0.1
1
10
RestingColitic
mR
NA
exp
ress
ion
rela
tive
to C
yclo
phili
n A
Ly6C+MHC+ [P2]
Cx3cr1 Cd163 Cd206 Tlr2 Tlr4 Ccr2 Il10 Il6 Tnfa0.1
1
10
RestingColitic
mR
NA
exp
ress
ion
rela
tive
to C
yclo
phili
n A
b F4/80+ Ly6CnegMHC+ [P3]
Cx3cr1 Cd163 Cd206 Tlr2 Tlr4 Ccr2 Il10 Il6 Tnfa0.01
0.1
1
10
100
RestingColitic
mR
NA
exp
ress
ion
rela
tive
to C
yclo
phili
n A
c F4/80+ Ly6CnegMHC+ CX3CR1hi [P4]
Supplementary Figure 8
Tlr1 Tlr3
Tlr5 Tlr6 Tlr7 Tlr9
Tlr2 Tlr4 A
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Ly6Chi [P1]
Ly6C+MHC+ [P2]
F4/80+ MHC+ [P3]
CX3CR1hi [P4]
TLR2
CD14
B
BMM P1 P2 P3 P40.01
0.1
1
10
mR
NA
exp
ress
ion
rela
tive
to C
yclo
phili
n A
BMM P1 P2 P3 P40.1
1
10
BMM P1 P2 P3 P40.001
0.01
0.1
1
BMM P1 P2 P3 P40.01
0.1
1
10
BMM P1 P2 P3 P40.001
0.01
0.1
1
mR
NA
Exp
ress
ion
rela
tive
to C
yclo
phili
n A
BMM P1 P2 P3 P40.01
0.1
1
10
BMM P1 P2 P3 P40.01
0.1
1
10
BMM P1 P2 P3 P40.01
0.1
1
10
Supplementary Figure 9