innate lymphoid cells in mucosal immunity and inflammation · 2014-10-22 · innate lymphoid cells...

Innate Lymphoid Cells in Mucosal Immunity and Inflammation

KAAACI-2012

Myoung Ho JANG

Laboratory of Gastrointestinal Immunology ,

WPI Immunology Frontier Research Center,

Osaka University

Division of Integrative Biosciences and Biotechnology, POSTECH

In the Gut,…

Commensal bacteria

Food proteins

Foods; approx. 150 g of protein per day (55 Kg/year) Commensal bacteria; 100 trillion (1014), approx. 1000 species, 1.5 Kg

Mucosal Tolerance

Active systemic non-response to antigen administered through the mucosal (oral/nasal) route

Day 1 Day 10 Day 24

Subcutaneous or Intraperitoneal

Antigen and adjuvant

Subcutaneous


Antigen

Antigen

Antigen

Subcutaneous Oral/Nasal

Immunity

Tolerance

Oral/Nasal



Subcutaneous or

Immunity Oral/Nasal

DTH, T cell proliferation, Antibodies and Cytokine

Mechanisms of oral tolerance (low-dose tolerance)

J Allergy Clin Immunol 2008;121:1344-50

Mechanisms of oral tolerance (high-dose tolerance)

J Allergy Clin Immunol 2008;121:1344-50

Diarrhea Model (food allergy); mast cell infiltration

Two sensitizations

14

i.p. OVA(50㎍) & alum (1㎎)

28 30 32 34 36 days

Intragastric challenges

0

i.g PBS or OVA(50㎎)

Jung-hwan Kim, POSTECH

Ivanov I. et al. Cell Host & Microbe. 4:337 (2008)

Ivanov I. et al. Cell . 139:485 (2009)

Ivanov I. et al. Cell . 139:485 (2009)

Segmented Filamentous Bacteria in the Intestinal Tract of Th17 Cell-Sufficient and Th17 Cell-Deficient Mice

21 JANUARY 2011 VOL 331 SCIENCE

Nature. 2011 May 12;473(7346):174-80

Mind-Altering Bugs (http://news.sciencemag.org/sciencenow/2011/08/mind-altering-bugs.html?ref=hp) Hundreds of species of bacteria call the human gut their home. This gut "microbiome" influences our physiology and health in ways that scientists are only beginning to understand. Now, a new study suggests that gut bacteria can even mess with the mind, altering brain chemistry and changing mood and behavior. In recent years, researchers have become increasingly interested in how gut bacteria might influence the brain and behavior, says John Cryan, a neuroscientist at University College Cork in Ireland. So far, most of the work has focused on how pathogenic bugs influence the brain by releasing toxins or stimulating the immune system, Cryan says. One recent study suggested that even benign bacteria can alter the brain and behavior, but until now there has been very little work in this area, Cryan says. To further investigate the mind-altering potential of benign bacteria, Cryan and colleagues at McMaster University in Canada fed mice a broth containing a benign bacterium, Lactobacillus rhamnosus. The scientists chose this particular bug partly because they had a handy supply and also because related Lactobacillus bacteria are a major ingredient of probiotic supplements and very little is known about their potential side effects, Cryan says. In this case, the side effects appeared to be beneficial. Mice whose diets were supplemented with L. rhamnosus for 6 weeks exhibited fewer signs of stress and anxiety in standard lab tests, Cryan and colleagues report online today in the Proceedings of the National Academy of Sciences.

Cryptopatches

(CPs)

Isolated Lymphoid Follicles

(ILFs)

iILF mILF

Bouskra D, et al. Nature. 456:507 (2008)

Peyer’s patches

(PPs)

CD11c RORg

B220

Solitary Intestinal Lymphoid Tissue (SILT) Isolated Lymphoid Follicle (ILF)

Follicle-associated epithelium (FAE)

Villus

Epithelial cells

Goblet cells

M-cells

Villous M-cell islands as M cell-independent gateway

Dense type

Diffuse type

Whole mount analysis Histological Analysis

UEA-1 WGA

AP+ (enterocyte)

AP-

(villous M cell)

Alcian blue (goblet cell)

Jang, Kweon et al., PNAS. 101, 6110 (2004)

M. Rescigno et al., Nat. Immunol. 2, 361 (2001)

M cell-independent antigen uptake routes

Chieppa M et al. J Exp Med. 203:2841 (2006)

DC extend their dendrite through the apical epithelium of villi

(CX3CR1-mediated) Niess J et al. Science. 307:254 (2005)

MA. Tam et al., Immunological Reviews 225:140 (2008)

Multiple antigen uptake routes in the gut

Gestational

Day 14

Gestational

Day 17

PP-null Mice

(1 wk)

PP-null/ILF-null Mice

(6 wk)

Blockade of signaling pathways

through LTbR

results in the lack of mucosal lymphoid tissues

LTbR- Ig

or

LTbR- Ig

Six time injections at weekly intervals

Control-Ig

-Treated

LTbR-Ig

-Treated

Mice Treated With LTbR-Ig in utero Lack Peyer’s Patches

Efferent lymphatic

Migration to MLNs

Naïve T cell

Small intestinal lamina propria

(LP)

Afferent lymphatic

M cell

Peyer’s patch (PP)

Follicle

Antigen presenting cells in the intestine

PP-DC Antigens Antigens

I can still induce oral tolerance!

PP-null mouse

pDCs

Macrophages

Eosinophils

CD11b-DCs CD11b+DCs

Multiple CD11c subpopulations are identified in the small intestinal LP

CD11b CD11c

Merge

CD

11c

CD11b

mPDCA

Jang et al., J. Immunol. (2006) Uematsu, Jang et al., Nat. Immunol. (2006) Uematsu, Fujimoto et al., Nat. Immunol. (2008)

Proposed roles of CX3CR1+ macrophages

Madcam-1 CD4+ T cells

Conversion to Foxp3+ T cells

CD103

Foxp3+ T cells

CD103

CD103↓

Maintaining Foxp3 & CD103

Lumen

Pathological Conditions (Few CX3CR1+ MФs)

CX3CR1+

MФs

Epithelial cells

Decreasing CD103+Tregs

Induction CD103+Treg

Mast cells

Eosinophils

ILCs; innate lymphoid cells

CD103+ DCs

CX3CR1+ macrophages

CD8+ T cells

Th17 cells

Tregs

IgA+ B cells

Basophils

All ILCs have morphological characteristics of lymphoid cells yet lack rearranged antigen receptors, a quintessential feature of adaptive immune cells. ILCs react promptly to a wide array of signals and serve important roles in lymphoid tissue formation, repair of damaged tissue, and tissue homeostasis, as well as in immunity against infectious microorganisms.

Hergen Spits et al., Annu. Rev. Immunol. 2011. 30:647.

Innate lymphoid cells (ILCs)

Cell Type Function Cytokines Major stimulating cytokines

NK cells (Cytotoxic ILCs)

Innate immunity against viral infections

Tumor immunosurveillance

IFN-Υ IL-18 IL-12 IL-15

*Rorgt+ ILCs Lymphoid tissue formation Tissue repair Innate immunity against

bacteria

IL-17 IL-22

IL-1b

IL-23

Type 2 ILCs (ILC2)

Innate immunity against extracellular parasites

IL-5 IL-13

IL-25 IL-33

Differential functions of ILC subsets

Hergen Spits et al., Annu. Rev. Immunol. 2011. 30:647.

*Rorgt+ ILCs, which express and require the nuclear hormone receptor retinoic acid (RA) receptor related orphan receptor (Ror) gt (encoded by the Rorc gene)

Spits H. & James P Di Santo (2011) Nat Immunol. 12(1):21

TH1

TH2

TH17

Treg

ILC1

ILC2

ILC17

ILC22

ILC-reg (?)

RORγt

NK cells

NH cells

MPPtype2 cells

Nuocytes

Ih2 cells

c-kit– sca-1+Thyhigh

NKp46+ cells

LTi-like cells

Unknown

T-bet

GATA3

RORγt

Foxp3

Id2

Id2

Id2 RORγt

Id2 RORγt

?

ILCs are the functional mirror images of T cells

Dr. Young-ae Lee

Lineage

Lineage negative

Sca-1

c-K

it

Lymphocyte gate

Small intestinal LP cells (BABL/c)

FSC-A

SS

C-A

Lin-c-Kit-Sca-1-

Lin-c-Kit+Sca-1-

A B

Lin-c-Kit+Sca-1- cells and Lin-c-Kit-Sca-1- cells were

sorted from small intestinal LP

Young-ae Lee, Unpublished

Lin-c-Kit-Sca-1- cells Lin-c-Kit+Sca-1- cells

Gene expression profiles of Lin-c-Kit-Sca-1- cells and

Lin-c-Kit+Sca-1- cells in small intestinal LP

Microarray:

GeneChip mouse Genome 430 2.0 Array chips (Affymetrix)

(Dr. Kumagai, PhD)

Biological process of up-regulated gene in Lin-c-Kit+Sca-1- cells compared to

Lin-c-Kit-Sca-1- cells

1

10

100

1000

10000

1

10

100

1000

10000

100000

1

10

100

1000

Immune regulation

Antimicrobial peptides Proteolysis

Re

lati

ve

mR

NA

le

ve

ls

(Lin

- c-K

it+S

ca

-1- v

s. L

in- c

-Kit

- Sc

a-1

- )

Lin-c-Kit+Sca-1- cells from small intestinal LP highly express

Il22, Csf2rb2, α-defensins, and mast cell proteases

Stephanie L. Sanos et al., Immunology. 2011, 132

Models for IL-22-instructed epithelial homeostasis

Charles L. Bevins et al., Nature Review. 2011, 9

Morphology and histology of Paneth cells

Paneth cells DEFA5 Stem cells (green)

Paneth cells (red)

Stem cells (green), Paneth cells (red) Crypt lumen

Immune regulation of intestinal barrier function

Simon P. Hogan et al., J Allergy Clin Immunol., 2009,124

Lineage

Lineage negative

Lymphocyte gate

Sl LP cells

FSC-A

SS

C-A

NKp46

c-K

it

CD4

LTi-like MCPs

ILC22

A

0 week 1 week 2 weeks 3 weeks 4 weeks

mIL-3 + mSCF

B

Lin-c-Kit+NKp46-CD4- cells can differentiate and proliferate

in the presence of IL-3 and SCF in vitro culture

A

0

10

20

30

40

50

Hprt1 Il22 Defa4 Defa5 Defa24 Defa-rs1 Defa-rs2 Defa-rs7

0

1

2

3

4

5

6

7

Rela

tive m

RN

A l

evels

(Ta

rget

gen

e v

s.

Hp

rt1

)

Lin-c-Kit+NKp46-CD4- (MCPs)

Lin-c-Kit+NKp46-CD4+ (LTi-like)

Lin-c-Kit+NKp46+ (NKp46+ ILC22)

Lin-c-Kit+NKp46-CD4- cells in the steady state

condition highly produce α-defensins, Mcpt2, Il17f, and Il22

FSC-A S

SC

-A

c-K

it

c-K

it

Isotype FcεRI

% o

f cell

nu

mb

er

0

1000

2000

3000

4000

5000

6000

7000

8000

9000



Lin-c-Kit+NKp46+ (NKp46 + ILC22)

mIL-3 + mSCF

C

D

*

*

*

**

0

10

20

30

40

50

60

IL-17E IL-21 IL-22 IL-23p19 IL-31 IL-33 CD40L MIP-3α




pg

/ml

Lin-c-Kit+NKp46-CD4- cells in the steady state

condition highly produce IL-17F and IL-22

IL-17 IL-21 IL-22 IL-31 IL-3 CD40L MIP-3α

*

0

500

1000

1500

2000

2500

3000

3500 Lin-c-Kit+NKp46-CD4- (MCPs)



pg

/ml

Lin-c-Kit+NKp46-CD4- cells highly produce IL-17F and

IL-22 protein in the stimulation with IL-1β or IL-23


LPMCs

BMMCs

mIL-3 + mSCF

LPMCs

BMMCs

Lin-c-Kit+Sca-1- -derived mast cells vs. bone marrow-

derived mast cells

Hprt1 Il22 Defa4 Defa5 Defa24 Defa-rs1 Defa-rs2 Defa-rs7

0

2

4

6

8

10

12

14

0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4

Rela

tive m

RN

A l

evels

(Ta

rget

gen

e v

s.

Hp

rt1

)

weeks

1.0 1.0 1.0 1.0 0.7 0.0 0.0 0.0

9.2

0.0 0.0 0.0

2.9

0.1 0.0 0.0 0.6 0.4 0.1 0.0

158.9

108.2

80.9

0.2 0.0 0.0 0.0 0.3 0.1 0.0 0.0

183.1

138.5

84.1

0

50

100

150

200

250

0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 0 2 3 4 weeks

Hprt1 Rorc Mcpt1 Mcpt2 Mcpt4 Mcpt5 Mcpt8 Mcpt10 Cpa3

Expressions of Il22, AMPs, and Mcpts are decreased according to

differentiation of Lin-c-Kit+NKp46-CD4- cells into mast cells

Allergic responses (IL-3 and SCF) Physiological conditions

IL-1β

Lamina propria

MCPT5

CPA3 IL-23 IL-22

IL-17F

α-Defensins

IL-22

IL-22

IL-17F

MCPs

ILC22

LTi

MCPs

MC

MC

Eo

MΦ

DC

Commensal

bacteria

MC

Summary I. CD4- LTi-like cells can constitutively produce α-defensins and mast cell proteases

under physiological conditions.

II. CD4- LTi-like cells produce the highest levels of IL-22 in the presence of IL-1β or IL-

23 compared with CD4+ LTi-like cells or ILC22.

III. CD4- LTi-like cells can differentiate into MCs in the presence of IL-3 and SCF. They

can be early mast cell progenitors, but can fully maturate compared with BMMCs.

Laboratory of Gastrointestinal Immunology, WPI, IFReC, Osaka University Dong-ju You Soo-jung Jin Zijin Guo Reiko Fukamizu Eriko Mano Young-ae Lee Laboratory of Host Defense, WPI, IFReC, Osaka University Shizuo Akira Department of Microbiology, College of Medicine, Ewha Womans University Yun-Jae Jung So-Yeon Woo Ju-Young Seoh

Laboratory of Gastrointestinal Immunology, IBB, POSTECH Min-seong Jang Jung-hwan Kim Chun-pyo Hong Eun-jung Lee Eun-ji Jeun Go-eun Lee Bo-su Jeong Bo-gie Yang Department of Mechanical Engineering, POSTECH Jun-sang Doh Ki-hean Kim

See you in Osaka!

Innate

lymphoid cells

(ILCs)

Lee YA, Unpublished data

Lineage

Lineage negative

Sca-1

c-K

it

Lymphocyte gate

Small intestinal LP cells (BABL/c)

FSC-A

SS

C-A

Lin-c-Kit-Sca-1-

Lin-c-Kit+Sca-1-

A B

Lin-c-Kit+Sca-1- cells and Lin-c-Kit-Sca-1- cells were

sorted from small intestinal LP

NK ILC2 LTi ILC22 ILC17

Lineage + - - (CD4+)

- -

Id2 + + + + +

CD117

(c-kit) +

(CD56 subset) + + + -

Sca-1 - + ? ? +

CD127

(IL-7R) + + + + +

RORγt - - (γc dependent)

+ + +

NK1.1

NKp46 + -

NK1.1 : -

NKp46 : + +/- -

Location Various FALC, MLN,

spleen, GALT

Fetal tissues,

Cryptopatch,

ILF

Mucosal sites

(LP, PP, MLN,

tonsil..)

Intestinal track

(colon)

Function Produce IFN-γ,

Cellular

cytotoxicity

Promote

TH2 cytokine

response

Formation of

PP, LN, ILF

IL-22 (pro-

inflammatory &

protective to

inflammation)

Produce IL-17

(proinflammatory)

Natural helper cell, Nuocyte, MPPtype2 cell, Ih2 cell

RORγtGFP/+ (C57BL/6–background) / Female / 7 wks (DOB : 2012.2.14)

FSC

SS

C

Lineage

Lin–

RORγt

NK

p46

Gated on

Lin–

CD4

Gated on

Lin–

RORγt

CD

4

ILC22

LTi

RORγt

Lin

ea

ge

NKp46

C-k

it

ILC22

CD4

C-k

it

LTi MCP

SI LP cells

ILC22

NKp46

Mast cells

Patho-physiological condition Physiological condition

Lin-c-

Kit+NKp46-

CD4-

Lin-c-Kit+

NKp46-CD4+

Lin-c-Kit+

NKp46+

IL-22

IL-17F

α-Defensins

Lamina

propria

Epithelial cells

IL-3 and SCF

MCPT5

CPA3

IL-22

α-Defensins Bacteria

Mcpt1

Mcpt2

Permeability

MMP-2, MMP-9

Inflammation

Eosinophils migration

MCPs

LTi ILC22

IL-23

IL-1β

Dual functions of Lin-c-Kit+NKp46-CD4- cells

as ILCs and as MCPs

IL-21

IL-22

IL-17F

IL-22

博多もつ鍋 (후쿠오카식 곱창전골)

http://www.daimaru.jp/image/1/cmdty/4152003_0300_L.jpg http://cfile234.uf.daum.net/image/15077D3D4D0C19070CD5EC

대한민국 양곱창 (韓国式ホルモン焼き)

Do these foods suppress intestinal inflammation through tissue-specific TOLERANCE ?

October in Finland (2006)

Cold water (4°C) “Tolerance”

1. Identification of villous M cells Proc Natl Acad Sci USA. 101:6110, 2004

2. New functions of intestinal DCs and eosinophils Nat Immunol. 7:868, 2006 J Immunol. 176:803, 2006 Nat Immunol. 9:769, 2008 J Immunol. 187:2268, 2011

3. Interation between DCs and Tregs in the gut J Immunol. 183:5608, 2009 Int Immunol. 20:307, 2008

4. Anti-inflammatory molecules in the gut (Atg16L1 and Semaphorin 7A) Nature. 456:264, 2008

J Immunol. 188:1108, 2012

Myoung Ho JANG, Ph D. Laboratory of Gastrointestinal Immunology, WPI Immunology Frontier Research Center, Osaka University

lamina propria

lumen

Foxp3 CD11c

Epithelium

Th17 cells

Tregs

CD103+ DCs

Eosinophils

CD11b+ DCs

Villous M cells Lumen

Peyer’s patch

Macrophages

Eosinophils

CD11b-DCs CD11b+DCs

CD11b

CD

11c

innate lymphoid cells in mucosal immunity and inflammation · 2014-10-22 · innate lymphoid cells...

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