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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 and adjuvant
Antigen
Antigen
Antigen
Subcutaneous Oral/Nasal
Immunity
Tolerance
Oral/Nasal
Antigen and adjuvant
Antigen and adjuvant
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
0 week 1 week 2 weeks 3 weeks 4 weeks
Lin-c-Kit+NKp46-CD4- (MCPs)
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
Lin-c-Kit+NKp46-CD4- (MCPs)
Lin-c-Kit+NKp46-CD4+ (LTi-like)
Lin-c-Kit+NKp46+ (NKp46+ ILC22)
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)
Lin-c-Kit+NKp46-CD4+ (LTi-like)
Lin-c-Kit+NKp46+ (NKp46+ ILC22)
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
0 week 1 week 2 weeks 3 weeks 4 weeks
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