Supplemental Material Supplemental Figure 1. Further Analysis of … · 2013-04-17 · ! 1! Supplemental Material Supplemental Figure 1. Further Analysis of CX3CR1 Expressing Myeloid

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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

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C

SiglecF

CX3CR1hi LP mφ [P4]

Resident Peritoneal mφ

M-CSF BM-derived mφ

e

P1 P2 P3 P5

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f

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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

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g

CX

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Resting flt3L0

20

40

60

80

100

120

Num

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Resting flt3L0

5

10

15

20

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Num

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CD

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DC

per

colo

n (x

105 )

Resting flt3L0

20

40

60

80

100

120

Num

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f Ly6

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(x10

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Resting flt3L0

20

40

60

80

100

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CX

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Non-injected control 3h 12h

[P1]

24h 48h

[P2]

[P3]

[P4]

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1.9 0.6 1.4 1.2 25.5

2.2 1.0 0.7 1.7 13.7

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B = Ly6C+MHC+ [P2]

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Gated: CD11b+ Ly6Gneg CD117neg

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monocytes

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Colitic

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IL10 eGFP

CD

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P1 P2 P3/P4 DC0

20

40

60

80

100

*** ******

% IL

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Cx3cr1 Cd163 Cd206 Tlr2 Tlr4 Ccr2 Il10 Il6 Tnfa0.01

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exp

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mR

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exp

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rela

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to C

yclo

phili

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b F4/80+ Ly6CnegMHC+ [P3]


0.1

1

10

100

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exp

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exp

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rela

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BMM P1 P2 P3 P40.1

1

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10


Documents

Supplemental Material Supplemental Figure 1. Further Analysis of … · 2013-04-17 · ! 1! Supplemental Material Supplemental Figure 1. Further Analysis of CX3CR1 Expressing Myeloid