Innate and Adaptive Immunity-Complementary & Distinct Host Defenses

Innate and Adaptive Immunity-Complementary & Distinct Host Defenses

INNATE ADAPTIVE

Hard-wired Requires Priming

Immediate Delayed or Immediate

Cell bound receptors Soluble and Cellular

Pattern-molecule recognition Antigen Specific

Macrophages, NK, non-prof. APC B-cells, T-cells

Different ligands selectively activate different TLRs

Different ligands selectively Different ligands selectively activate different TLRs activate different TLRs

TLR

2TL

R2

TLR

4TL

R4

TLR

5TL

R5

TLR

6TL

R6

TLR

9TL

R9

TLR

3TL

R3

TLR

1TL

R1

TLR

7TL

R7

TLR

8TL

R8

TLR

11TL

R11

FlagellinFlagellin

LPS LPS

CpG DNACpG DNALipoproteinsLipoproteins

ds RNAds RNAss RNAss RNA ProfilinProfilin--likelike

ProtozoanProtozoan

TLRs are expressed by many cell types throughout the gastrointestinal tract

TLRs are expressed by many cell types throughout the gastrointestinal tract

IEC (IEC (stomachstomach, SI, , SI, coloncolon))MonocytesMonocytes//macrophagesmacrophagesDendriticDendritic cellscellsMyofibroblastsMyofibroblastsEndothelialEndothelial cellscellsAdipocytesAdipocytes

TLR4TLR4

CarioCario et al. Am J et al. Am J PatholPathol 2002 160:1652002 160:165

IECIEC

apicalapical

In constrast, TLR5 is preferentially expressedat the basolateral pole of differentiated IEC

In constrast, TLR5 is preferentially expressedat the basolateral pole of differentiated IEC

SubmucosaSubmucosa

LumenLumen

J. Immunol. 2001 167:1882J. Immunol. 2001 167:1882

Infect. Immun. 2000 68:7010Infect. Immun. 2000 68:7010

T84T84

ColonColontissuetissue

Cell

Nucleus

TLRs induce multiple signal transduction pathwaysvia different adaptor proteins

TLRs induce multiple signal transduction pathwaysvia different adaptor proteins

resident resident microfloramicroflora

Mechanisms of intestinal epithelial ”TOLLerance”

Mechanisms of intestinal epithelial ”TOLLerance”

Decreased Decreased surface expressionsurface expression

IntestinalIntestinalEpitheliumEpithelium

NFNFκκBB

Ligand neutralizationLigand neutralization

Mucins DefensinsMucins DefensinsIgA TrefoilIgA Trefoil

Receptor localizationReceptor localizationState of differentiationState of differentiation

TLR4TLR4

NormalNormal

ST2ST2 TollipTollip

Signaling suppressorsSignaling suppressors

PPARPPARγγ

TGFTGFßß SOCSSOCS

TRIADTRIAD3A3A

CytokineCytokine

Intestinal epithelial TLR4 expression is significantly increased in IBD

Intestinal epithelial TLR4 expression is significantly increased in IBD

HealthyHealthy

CrohnCrohn’’ss DiseaseDisease

Ulcerative ColitisUlcerative ColitisCario, Podolsky. Infect Immun 2000;68:7010Cario, Podolsky. Infect Immun 2000;68:7010

TLR4 alteration associated with IBDTLR4 alteration associated with IBD

Upregulation of mucosal TLR4 expression in active, human IBD

Association of TLR4D299G-polymorphism with IBD in selective populations

Murine TLR4-“gain-of-function”: spontaneous intestinal inflammation with TH1-excess

Murine TLR4-“loss-of-function”: increased susceptibility to DSS-colitis and gram─ infections

Flagellins stimulate aberrant intestinal immune reactions via TLR5 as dominant antigens in colitis Flagellins stimulate aberrant intestinal immune

reactions via TLR5 as dominant antigens in colitis

Lodes et al. JCI 2004;113:1296Lodes et al. JCI 2004;113:1296

••Marked Marked TT--cellcell reactivityreactivity againstagainst flagellinflagellin••FlagellinFlagellin--specificspecific T T cellscells induceinduce colitiscolitis in SCID in SCID micemice••SerologicalSerological responseresponse to to specificspecific flagellinsflagellins in CD in CD ptspts..

TLR4 mutant and knockout mice are highly susceptible to develop DSS colitis

TLR4 mutant and knockout mice are highly susceptible to develop DSS colitis

Fukuta et al. Am J Physiol GI 2005;288: G1055

4.154.15±±0.790.79C57BL6/JC57BL6/J

7.187.18±±0.560.56C3H/HeJC3H/HeJ

DSS colitis scoreDSS colitis scoreMouse strainMouse strain

Mahler et al. Am J Physiol GI 1998;274: G544

RakoffRakoff--Nahoum et al. Nahoum et al. Cell 2004;118:229Cell 2004;118:229

DSS 2%, 7 days, F2DSS 2%, 7 days, F2

Basal TLR activation may be protective against acute injury through its beneficial effects in the intestinal epithelium

Basal TLR activation may be protective against acute injury through its beneficial effects in the intestinal epithelium

EpitheliumEpithelium

Acute injuryAcute injuryintestinal epithelial barrierintestinal epithelial barrier

Com

men

sals

Com

men

sals

Healing ↑Barrier protection ↑Tolerance ↑

TLR

TLR2 assists the host to confer intestinal epithelial barrier function

TLR2 assists the host to confer intestinal epithelial barrier function

0

50

100

150

200

0 15 30 45 60 75 90 105 120

Time (mins)

TER

(% o

f neg

ativ

e co

ntro

ls)

TLR2DN TLR4DN

+ PCSK* *

# #

°°

TLR4DNTLR4DN-- vs. TLR2DNvs. TLR2DN--transfected monolayers: transfected monolayers: *p<0.01; #p<0.001; *p<0.01; #p<0.001; °°p<0.05p<0.05

Cario et al. Gastroenterology 2004;127:224Cario et al. Gastroenterology 2004;127:224

XX--YY

ZOZO--11-- PC

SKPC

SK

ZZ

+ PC

SK+

PCSK

XX--YY

ZOZO--11

ZZ

IECIEC

Commensal compounds assist the host to confer intestinal epithelial barrier functionCommensal compounds assist the host to confer intestinal epithelial barrier function

Madsen, et al. Madsen, et al. Gastroenterology 2001;121:580Gastroenterology 2001;121:580

T84

Otte, Podolsky. Am J Physiol GastrointestOtte, Podolsky. Am J Physiol GastrointestLiver Physiol 2004;286:G613Liver Physiol 2004;286:G613

+LPS 10ng/ml+LPS 10ng/ml MMθθ CD4+ T cellsCD4+ T cells

controlcontrol TLR4TLR4--//-- TLR4/Stat3TLR4/Stat3--//--

in thein the absence of absence of TLR4, the onset of TLR4, the onset of chronic chronic enterocolitisenterocolitisis delayed,is delayed, and and activity of colitis is activity of colitis is improvedimproved

Kobayashi et al. JCI 2003;111:1297Kobayashi et al. JCI 2003;111:1297

Commensal-mediated TLR4 dysfunction contributes to the development of TH1-intestinal inflammation

Commensal-mediated TLR4 dysfunction contributes to the development of TH1-intestinal inflammation

Com

men

sals

Com

men

sals M

ucosaM

ucosaInvasion

TLRTH1TH1--typetype

CytokinesCytokines

Aberrant TLR4/MyD88 Aberrant TLR4/MyD88 signalingsignaling may may favorfavor chronicchronicintestinal inflammation through intestinal inflammation through commensalcommensal--mediated mediated

proinflammatoryproinflammatory TH1TH1--effects in the lamina effects in the lamina propriapropria

Injury ↑Barrier destruction ↑Intolerance ↑Chronic inflammatory diseaseChronic inflammatory disease

Therapeutic Exploitation of the InnateImmune System

Activation?

Inhibition?

Innate Immune Responses are Mediated by Several Receptor Classes

• TollToll--like receptorslike receptors

• NodsNods

• Cd1

• Other

Mutations in LRRs of NOD2 link tothe risk of Crohn’s disease

Gene

Protein

NBD LRRCARD1 1040

R702W

G908R

3020insC

0

20

40

60

80

1 0 0

1 2 0

1 4 0

1 2 3 4

100

80

60

40

20

0

120

140CFU % vs Caco2

***

***

p<0.05 vs Caco2

p<0.05 vs MOCK

CD-mutant Nod2 exhibits defective epithelial killingof intracellular invasive bacteria

NOD2 is membrane associated

- - E A L L Q A L E R N D T I L E V W L R G N T F S L E E V D K L G C R D T R L L L

NOD2insC3020

Amino acid sequences of the C-terminal domain of NOD2 and NOD2 3020insC mutant associated with Crohn’s disease.

IDENTIFICATION OF GRIM-19 AS NOD2 INTERACTING PROTEIN

COS7

/MO

CK+

Xp-G

RIM

19

COS7

/Fla

g-NO

D2+

Xp-G

RIM

19

GRIM19

NOD2

GRIM19

IP anti-NOD2WBanti-GRIM19

anti-NOD2

WBanti-GRIM19(anti Xpress Ab)

1. Immunoprecipitation 2. Immunostaining

Merge

XpGRIM19

GFP-NOD2

GRIM-19 ACTS DOWNSTREAM OF NOD2 AND IS REQUIRED FOR NF-kB ACTIVATION

0

20

40

60

80

100

120

- MDP

+ MDP-LD

NOD2 1 nggrim-19 siRNA-1control grim-19 siRNApSUPERgrim-19 mRNA level

----1

+---ND

++--0.32*

+--+0.98

*

p=0.027

Rel

ativ

e lu

cife

rase

act

ivity

+-+-0.89

p=0.218

Crohn’s Disease is Associated with Reduced Early Inflammatory Response to Subcutaneous Injection of E coli

Crohn’s Disease is Associated with Reduced Early Inflammatory Response to Subcutaneous Injection of E coli

Marks et al, Lancet, 2006

Healthy ControlHealthy Control

CrohnCrohn’’s s DiseaseDisease

Nod2/Card 15 Reduce TLR-2 mediated IL-12 and IFN Production

Nod2/Card 15 Reduce TLR-2 mediated IL-12 and IFN Production

Watanabe et al, Nature Immun, 2004

Multiple Stimuli Converge on a Common Signaling Pathway in IBD

MDPNod 2

LPS/PGN

TLR 4/TLR2

Myd88

NIK

IKK

NF-κB

ILs/CC

Apoptosis?

TNF/TNFR

IL1/IL1R

OCTN1?DLG5?

Nicolas Barnich Hans Christian ReineckerElke Cario Ramnik XavierTakakazu HisamatsuJan-Michel Otte Beth McCormickManabu SuzukiTomo TeradaJesus Yamamoto-FurushoJose AguirreKathryn Devaney