mononuclear phagocytes - origins, fates & functions - … · clp mep granulocytes m ... 11 •...

Mononuclear Phagocytes- Origins, Fates & Functions -

Dr. Steffen Jung

1The screen versions of these slides have full details of copyright and acknowledgements

1

Dr. Steffen JungDepartment of Immunology

The Weizmann Institute of Science Rehovot, Israel


2

The mononuclear phagocyte systemVan Furth and Cohn (1968)

Macrophages

Ilya (Eli) Metchnikoff(1845-1916)

• Role in immunity • Homeostasis (physiological inflammation)

Nobel prize 1908

Dendritic cells

Ralph Steinman(1943-2011)

Nobel prize 2011

• Unrivalled migratory • Naïve T cell stimulators

3

• Macrophages

• Dendritic cells

• Monocytes

The mononuclear phagocyte system (2)Van Furth and Cohn (1968)

Outline of presentation:

• Mononuclear phagocyte development

• Mononuclear phagocyte functions in the intestine


Dr. Steffen Jung


4

HematopoeticStemCell

Myeloid lineage

Lymphoid lineage

CommonMyeloidPrecursor

CommonLymphoidPrecursor

MONONUCLEAR PHAGOCYTES

GRANULOCYTES(PMN)

T CELLS

B CELLS

NK CELLS

LYMPHOCYTES

MONOCYTES

MACROPHAGES

DENDRITIC CELLS, DC

The hematopoetic system

5

2005

Monocytes

Varol et al., 2007

GFP

Mononuclear phagocyte originsCX3CR1

GFP

CMP MPHSC

CLP MEP Granulocytes

MΦDCProgenitor

Lin-

CD34+

ckit+CD16+

CX3CR1+

CX3CR1GFP

6

blood

Ly6C+

Ly6C-

Monocytes

CD

115

CX3CR1/GFP

Ly6C

/G

MouseMonocyte subsets

Geissmann, Jung & Littman, 2003

CX3CR1/GFP

CX3CR1GFP


Dr. Steffen Jung


7

CD115+ blood cells

Cros et al., 2010

Ziegler-Heitbrock et al., 1996

Human monocyte subsets

CD14++ CD16+/- CD14dim CD16++

Ly6C+ Ly6C-

Human

Mouse

Monocyte subsets (2)

8

blood

Monocytes

Ly6C-

“Inflammatory”monocytes

Auffray et al., 2009

“Patrolling”monocytes


CCR2+

Ly6C+

Monocyte subsets (3)

9

Blood

Ly6C+

Monocytes

Varol et al.,2007

Fogg et al.,2006

MΦDCPrecursor


Ly6C-?

Bone marrow

• 2 alternative routes to Ly6C- monocytes Sunderkotter et al., 2007 Yrlid et al., 2006Varol et al., 2007 Ly6C+

Monocyte origins• Which pathway dominates in steady state?

• Ly6C+ but not Ly6C- monocytes express CCR2CCR2+CCR2+

• CCR2 is required for exit of Ly6C+

monocytes from BMSerbina et al., 2006; Shi et al.. 2011


Dr. Steffen Jung


10

Blood

Ki-Wook KimYochai Wolf

CD45.2CD115

CD

135

CD

117 MDP

% o

f Max

FSC

• Monocyte precursors (MDPs) equally represented from genotypes

BM

• Lymphocytes equally represented from both genotypes

Mixed BM chimeras

Monocyte origins (2)

CCR2-/- (CD45.2) BMWT (CD45.1) BM

B cells

CD45.2

52.2 1.54

46.20.12

CD

45.1

Gr1

B220

CD

115

11

• Both monocyte subsets equally affected by CCR2 deficiency

CCR2-/- (CD45.2) BMWT (CD45.1) BM

Mixed BM chimeras

Monocyte origins (3)

Ly6C-

CD45.2

87.9 0.325

11.70.0464

CD

45.1

CD

45.1

CD45.2

84.5 0.375

15.10.0536

Ly6C+

CX3CR1 GFP

Ly6C

Monocytes

Wt

/ CC

R2-/-

ratio

BM

MP MDP Ly6C+ Ly6C- Ly6C+ Ly6C- Ly6C+ Ly6C-

monocytesspleenblood

0.0

0.5

1.0

1.536

9

12

1518

21

12

Blood

Ly6C+

MonocytesMΦDCPrecursor

Ly6C-

• Steady state monocytes form a developmental series

Bone marrow

Conclusion IMononuclear phagocyte origins


Dr. Steffen Jung


13

Blood

Ly6C+

Monocytes

Ly6C-

Monocyte fates

14

Diphtheria Toxin (DTx)

DTxReceptor

(DTR)

DTA

• Mice are naturally DTx resistant because they lack high affinity DTx receptor (hbEGFR)

• Primate DTR transgene confers DTx sensitivitySaito et al., Nat. Biotech. 2001 Jung et al., Immunity 2002

Conditional mononuclear phagocyte ablation

CD11c Promoter

DTR GFP

Ablation of :• CD11c expressing cells

• All CD11chigh cDCs

• Defined macrophage populations

CD11c-DTR mice

+ DTx

15 Varol et al., JExMed 2007

Mononuclear phagocyte originsIntestine without graft

monocytes + DTx

in vivodifferentiation

• Monocytes reconstitutemacrophages

CD11c-DTR mice

Intestine with graft

CX3CR1GFP


Dr. Steffen Jung


16

DonorDC-depleted

mouse

CD

11c

GFP

CD

11c

GFP

CD11chi

cDCs

* gated on CD45.1+ cells

Monocyterecipient

CD

11c

GFP

in vivodifferentiation

MDP

MDPrecipient

GFP

CD

11c

• MDPs, but not monocytes yield classical splenic cDCs

Mononuclear phagocyte origins (2)

+ DTxMonocytes

CD11c-DTR mice

Varol et al., JExMed 2007

17

• MDP are absent from circulation

• Identification of circulating dedicated cDC precursors, pre-cDCLiu et al., Science 2009

• Adoptive transfers

MDP

mono Pre-cDC

cDC

Mononuclear phagocyte origins (3)

MDP

PrecDC

B22

0

CD

11b

CD11c CD11c

% D

onor

-der

ived

cel

ls

18

monocytes

Ly6C+Ly6C-

monocyte-derivedMacrophages

“MoDCs”

BM

MP

MDP

HSCYOLK SACPrimitive

macrophages

FETALLIVER

Macrophages

E7.5-8.5

Pre-natal

Selfrenewal

Pre-DC

CDP

PDC

CD4+ CD8α+

CD103+DN

FLT3-dependent classical DCs

ThroughAdulthood

Conclusion II: Mononuclear phagocyte origins

Selfrenewal

Granulocytes


Dr. Steffen Jung


19

Gastrointestinal tract

• Breakdown of balance

Inflammatory Bowel Disease (IBD)

Tolerance> Commensals

Defense> Pathogenic

enterobacteria

• Bioreactor for food breakdown (>1013 bacteria)

• Large surface & thin border(single cell epithelial layer)

• Challenge to the immune system

Colon1011-12

CFU/ml

20

CX3CR1GFP

CX3CR1GFP

Intestinalmacrophages

21

CX3CR1 CX3CL1Fractalkine

CX3CR1

GFP

Jung et al., MCB 2000Kim et al., BLOOD, 2011CX3CR1GFP

mucin stalk CX3C

45 kb

ATG TM

• Targeted replacement of chemokine receptor with GFP reporter prominent expression in mononuclear phagocytes

• BAC transgene encoding reporter under CX3CL1 promoter control

The CX3CR1/L1 reporter animals

CX3CR1GFPCX3CR1GFP:CX3CL1cherry

: CX3CL1Cherry mice


Dr. Steffen Jung


22

MacrophagesEpithelial/ Goblet cells

CX3CR1GFP:CX3CL1cherry

Ki-Wook KimBlood 2011

23

Analysis of colon of CX3CR1gfp mouse 87 %

2.5 %

82 %

82 %

Gated on living CD45+ cells

MH

C II

(I-A

b )

FSC CD11b

CD

11c

CX3CR1 / GFP

CD

103

CD103+CD11b-

CD103+CD11b+

CX3CR1+ CD11b+

CX3CR1- CD11b+

CD11c+

DCs

MΦ’s

R1 R2

R3

R1CD11c+

CD11b-

R2CD11c+

CD11b+

R3CD11c-

CD11b-

Intestinal lamina propriamononuclear phagocyte compartment

Zigmond

24

CX3CR1+ macrophagesmonocyte-derived

Varol, Zigmond & Jung, Nature Reviews Immunology 2010

Varol, C., et al.Bogunovic, M. et al., Immunity 2009

Intestinal lamina propriamononuclear phagocyte compartment (2)

CD103+ Dendritic cellspreDC-derived

CX3CR1+ Macrophagesmonocyte-derived


Dr. Steffen Jung


25

naïve CD4T cell

priming site

Treg

Th17

Th2

Th1

LAM

INA

PR

OP

RIA

Msn

LN

Effector site

(Maintenance selective

expansion)C

CR

9, a

4b7

Th1Treg

Th2

Th17

CC

R7

Task division between migratory DCs and resident MFs

CD103+

dendritic cells

CX3CR1+

macrophages

Innate lymphoid

cells

LTi

gd

NHC

steady state

Scheme compiled from data of Pabst, Agace, Kelsall, Mowat, Powrie and Pulendran groups25

26

Steady state

CD

11b

Cx3cr1/GFP

Resident MFs

Ehud Zigmond

Inflammation• Exposure to dextran sodium sulfate

(DSS) in drinking water for 7 days

Lesions in epithel

27

CD

11b

Cx3cr1/GFP

Resident MFs

Inflammation recruits a novel CX3CR1int

cell subset

day 4 day 7

Inflammation

CD

11b

Cx3cr1/GFP

See also:Weber et al., EJI 2010, Platt et al., JI 2010 Rivollier et al., JExMed 2012 Ehud Zigmond

Steady state

% o

ut o

f CD

45+

livin

g ce

lls

0 5 10 15 200

10

20

NeutrophilsCX3CR1dimF4/80lowCX3CR1highF4/80high


Dr. Steffen Jung


28

CX3CR1int infiltrate is monocyte-derived

day 0 2 4 5 6 7 DSS 2% in drinking water

Analysis

day 3

• Ly6Chi monocytes seed tissue, give rise to Ly6Clo cells

MHC II

CD

11c

day 1

• Cells gain CCR7 expression

Ehud Zigmond

Monocyte transfer

F4/80

Ly6C

CX3CR1/GFP

CD

45.1

Monocyte recipient

w/o graft

Ly6C

CX3CR1/GFP

CD

45.1

F4/80

CD

11c

MHC II

29

CX3CR1/GFP CD45.1

Mixed monocyte recipient

CD

45.1

Mixed monocyte graftCCR2 KO WT

CD45.1

• Only WT cells but not CCR2 deficient cells seed inflamed tissue

CX3CR1int infiltrate is monocyte-derived, requires CCR2

Monocyte transfer


Analysis

Ehud Zigmond

30


Anti-CCR2 ab Monocyte depletion

DSS + MC21

DSS only

Ly6C

CD

11b

CX3CR1int monocyte-derived infiltrate promotes inflammation

Ehud Zigmond


Dr. Steffen Jung


31

Expression analysis of monocyte progeny during steady state and inflammation

Resident MFs Infiltrating MFs Monocytes

Steady state DSS Colitis

Ly6C+ monocytesCX3CR1hi resident macrophages

CX3CR1int infiltrating macrophages DSS Colitis

Steady state

Ehud Zigmond

• CX3CR1hi resident MFs express IL-10 (Pabst, Kelsall, Pulendran)

• CX3CR1int MFs express pro-inflammatory cytokines (IL6, IL23)

fold

cha

nge

CX

3CR

1int o

ver C

X3C

R1hi

QRT-PCR

IL6 IL23 IL10 TNFa-16-12-8-4048

1216

32

TLR-2

PDGiE-

DAP

MDP

NOD1

NOD2

RIP2

CARD9

IKKa

IKKg

IKKb

IkB

NFkB IL-6

• Expression of recruited CX3CR1int cells over resident CX3CR1hi cells

TLR2 & NOD-like receptor signaling pathway

CX3CR1int MFs respond to bacterial products

Ehud Zigmond

33

• Infiltrating MFs, but not resident MFs secrete IL-6 in response to TLR2 / NOD2 ligands

in vitro assay

Pam3

Muramyl-di-peptide

CX3CR1int MFs respond to bacterial products (2)

Ehud Zigmond


Dr. Steffen Jung


34

CD103+ DCs CX3CR1+

MF’s

Conclusion

Ly6C+

“Education”

Homeostasis

IL10IL10

• Monocytes give rise to CX3CR1+ macrophages in lamina propria

• Are in steady state educated by environment

• Under inflammation monocyte infiltrate fails to be “tamed” respond to bacterial product and exacerbate disease

LN

IL6IL6

Inflammation

LN

CC

R 7

34

35

Simon Yona

Ki-wook Kim

Ehud Zigmond

Diana Rashkovan

Yochai Wolf

Tegest Aychek

Alexander Mildner

Rita Krautgammer

Chen Varol

Tel Aviv-SouraskyMedical Center

Dan LittmanSkirball InstituteNYU Medical Center

Frederic GeissmannKing’s College, London

36

mononuclear phagocytes - origins, fates & functions - … · clp mep granulocytes m ... 11 •...

Documents