Molecular mechanisms of bacteria-hostinteractions at the epithelial cell level
Chair for BiofunctionalityDepartment of Food and Nutrition Science
Department of Medicine(www.wzw.tum.de/bflm)
Technical University of MunichProf. Dr. Dirk Haller
Robert Koch (1843 – 1910)
Theory about germs that cause infectious disease (1876)
Disease due to infectious microbes
Rudolf Virchow (1821 – 1902)
Theory about the cellular pathology of the diseased organ (1858)
Disease due to organ failure
Lessons from the past: germ theory vs. organ pathology
Normal gut
Ulcerative colitis
Crohn ׳s disease
Environmentaltriggers
Geneticpredisposition
• CARD15, NCF4, ATG16L1• TNFSF15, PTPN2, IL-23R
Microbial defenseImmune function
• ABCB1, PTGER4, SLC22AEpithelial function
Etiology of chronic inflammatory bowel diseases (IBD):a paradigm for bacteria-host interactions
Microorganisms in the gut (Dysbiosis) •
Infections, Smoking, Diet, NSAIDs •
Normal Gut
Enteric bacteria(SPF)
Enteric bacteria(SPF)
Wild type Wild type
Enteric bacteria(SPF)
Enteric bacteria(SPF)
Bacteroidesvulgatus
IL-10-/-
Enterococcusfaecalis
Chronic Inflammation
HLA-B27 IL-10-/- HLA-B27
Bacteroidesvulgatus
Enterococcusfaecalis
Enteric bacteria are not all equal in their capability to trigger chronic intestinal inflammation
Kim et al. 2007 Inflam. Bowel Dis. and 2005 Gastroenterology
Normal gutChronic
inflammation
SIgA(Tr1) IL-10
(Th3) TGF-βIL-11IL-15
IL-12 - IFN-γ (Th1)IL-23 - IL-17 (Th17)IL-13 (Th2)Smad7
Protective Colitogenic• Flagellin• GelE
CpG DNA •p40/70 •
RelA
U UUPP
Defensinesand
mucus
Epithelial cellintegrity
Failure of the epithelial cell homeostasis facilitates the loss of barrier function and chronic inflammation
Microbial and nutritional
signals
Diseasemechanisms
Ligand
Receptor
Signaltransduction
Gene- and protein-expression
Cell isolation
Cellular extractand fractions
Protein
2D-PAGEand
MALDI-TOF MS
Proteomeanalysis
Interface function of intestinal epithelial cells: from the proteome analysis to molecular mechanisms
Hostsignals
Clavel and Haller 2007 Inflam. Bowel. Dis.Werner and Haller 2007 Mutation Res.
What are the molecular mechanisms of bacteria-host interactions
Inflammation meets metabolism: cellular stress in the pathogenesis of chronic inflammation
Microbial determinants for pro-and anti-inflammatory effects at the epithelial cell level
IL-10-/-
IL-10-/-
Wild type
E. faecalis
Germ-free
Protective effects
Signalintegration
Colitogenic effects
Germ-free (sterile) mouse models for chronic intestinal inflammation: modeling bacteria-host cross-talk
Wild type
Germ-free
E. faecalis
IP-1
0 m
RN
A e
xpre
ssio
n(fo
ld in
crea
se)
0
5
10
15
20
25
GF 1 14 GF 1 14 time (weeks)
Histopathology(Score ± SD) Distal colon
E. faecalis OG1RF− + + − + +
0.5(0.1)
3.6(0.2)
Wild type IL-10-/-
0.6(0.1)
0.8(0.1)
0.3(0.1)
0.2(0.1)
*
*
*
Intestinal epithelial cell activation at early and late stages of bacterial colonization in wild type and IL-10-/- mice
Ruiz et al. 2005 J. Immunol.
TGF-βR2TGF-βR1
Enterococcus faecalis
TLR2
IP-10NF-κB
CBP/p300
P-Ac-Histone
IKK-β
IκB/NF-κBP-Smad2/3
TGF-β
Smad4
Ruiz et al. 2005 J. Immunol.
2
Haller et al. 2003 J.BC
1
Haller et al. 2002 JBC
15-deoxy-Δ12,14-prostaglandin J2
PP2A
3
Ruiz et al. 2004 JBC
Host-derived anti-inflammatory feed-back mechanisms in epithelial cells: signals from a complex environment
Inflammation
Time
Infla
mm
atio
n
Homeostasis
Chronic inflammation
Healing
Chronicinflammation
Enteric bacteria
Microbial-host cross-talk: from physiological inflammation to chronic inflammation
time post inoculation
Histoscore (±std) L.reuteri count (cfu) 1 x 108 per gram
Ileum Jejunum Ileum RLF-mice 0,7 (±0,24) - - 2d RLF-mice + 0,5 (±0,0) 3,9 5,4 RLF-mice 0,7 (±0,24) - - 6d RLF-mice + 1,1 (±0,2)* 6,5 7,8 RLF-mice 0,38 (±0,13) - - 21d RLF-mice + 0,55 (±0,24) 6,7 7,9
Reconstituted lactobacilli-free (RLF) mice as a model of bacteria-host interaction in the presence of a microbiota
J. Nutr. 2008
inoculation 2 6 21Num
ber o
f reg
ulat
ed /
iden
tifie
d pr
otei
ns
0
2
4
6
8
10
12
days
pH 3 pH 10
MasskDa
100
15
25
50
pH 3 pH 10
6 days after inoculation with L.reuteriCTRL
6
78
5 5
3
4 4
3
1
2 2
1
8 7
6
pH 3 pH 10
MasskDa
100
15
25
50
pH 3 pH 10
6 days after inoculation with L.reuteriCTRL
6
78
66
7788
5 55 5
3
4 4
333
44 44
33
1
2 2
111
22 22
11
8 7
6
88 77
66
β-Actin
I-FABP
grp58
- + + + L. reuteri2 6 21 days after inoculation
2 days 6 days 21 days 1 2 days 6 days 21 days 2 2 days 6 days 21 days 3 2 days 6 days 21 days
fold
cha
nge
-20
-10
0
10
20
30
40
50 IL-6 IL-1α MIP-2 A20
L.reuteri L.reuteri L.reuteri L.reuteri
**
**
*
*
IL-6 IL-1β MIP-2 A20
Reconstituted lactobacilli-free (RLF) mice as a model of bacteria-host interaction in the presence of a microbiota
What are the molecular mechanisms of bacteria-host interactions
Inflammation meets metabolism: cellular stress in the pathogenesis of chronic inflammation
Microbial determinants for pro-and anti-inflammatory effects at the epithelial cell level
Gel-basedprotein separation
1541
.868
1649
.903
1086
.862
1459
.719
1776
.714
1034
.818
1579
.791
2836
.480
1420
.752
1907
.632
2063
.611
1224
.763
832.
510
1148
.731
1352
.755
934.
586
2209
.628
0
1
2
3
4
5
6
4x10
Inte
ns. [
a.u.
]
1000 1250 1500 1750 2000 2250 2500 2750 3000m/z
Inte
nsity
m/z
2D-SDS-PAGE
Quantification and identification of protein expression changes in complex samples: from health to disease
Spot intensityProteolyticdigestion
Mass spectrometry (MS)
MALDI-TOFESI
WT
IL-10-/-
A Proteome analysis
× 3,6Grp-78
B Western Blot
Grp-78
β-actin
IL-10-/-WT
× 4,6
Induction of the ER stress response protein (Grp)-78 in the epithelium: from animal models to human IBD
Shkoda et al. 2007 Gastroenterology
Grp-78
β-actin
NI I
Western Blot
C Validation in IBD patients
NI I
ATF-6IRE-1α/β
PERK
Grp-78
Ribosome
Endoplasmic reticulum stress
+ ADP
S-S
Endoplasmic reticulum (ER) stress contributes to the pathogenesis of chronic degenerative diseases
ApoptosisAdaptation
Chronic inflammation
Epithelial cells are susceptible for ER-associated stress mechanisms in IBD: what is the evidence?
Bertolotti et al. 2001 J. Clin. Invest. ER-stress increased sensitivity to DSS colitis in IRE1β-deficient mice
Shkoda et al. 2007 GastroenterologyATF-6-mediated ER-stress in the epithelium of IL-10-deficient mice and IBD patients: protective role for IL-10
Heazlewood et al. 2008 PLOS MedicineAbberent mucin assemly causes ER stress-associated colitisand goblet cell loss in the epithelium
Kaser et al. 2008 CellXBP-1 variants are associated with ER-stress in Paneth cells and human IBD
Nucleus
TNF
RelA
P P PRelA
NF-κB
Inflammation
U
Bacteria
TNFR
U UP
P
P
TLR IKKα/β/γ
Stress responseATF6 XBP1s CHOPAP-1
UPRE
IL-10 IL-10
mtUPR
ROS
Cpn60
Cpn60
Cpn60
ATF6
Grp-78
IRE-1
PERKUPR
Cellular mechanisms for the regulation of stress responses: inflammation meets metabolism
17/9* 8/4* 38/38*
CRC UC UCCD
N=6 N=6 N=6 N=2
3/3*
Differential protein expression profile in primary IEC from colon and ileum of IBD patients: a pilot study
Shkoda et al. 2007 J. Proteome Res.
Non-inflamed (NI)
Inflamed (I)
Up
26 12UC Patient (No 1)
UC Patient (No 2)
Down
3 0
Comparison of inflamed versus non-inflamed tissue regions: the better-matched controls?
Highly interrelated protein networks indicate metabolic changes in primary IEC under chronic inflammation
Tissue pathology
Protektivesignals
Inflammatorysignals
Homeostasis
• Increase in glycolosyis• Increase in β-oxidation• Increase in apoptotic mechanisms• Increase in anti-oxidative responses• Increase in cell stress responses• Decrease in mitochondrial function
Shkoda et al. 2007 J. Proteome Res.Werner et al. 2007 J. Proteome Res.
Bibliometric co-citation analysiswith 66 proteins
Inflammatory Bowel Diseasesand Colorectal Cancer
Autoimmunityand Allergy
NeurodegenerativeDiseases
Obesity, insulin resistanceand Type 2 Diabetes
Inflammation meet metabolism: common disease mechanisms for chronic degenerative pathologies?
Inflammatory stressresponse
Metabolic stressresponse
Elias Metchnikoff
Gut bacteria contribute to diseasedevelopment (1907)
Disease due to microbial dysbalance
Rudolf Virchow
Theory about the cellular pathology of the diseased organ (1858)
Disease due to organ failure
Lessons from the past may be valid in the future
University of ZürichGerhard Rogler
FundingDFG, BMBF, Danone StiftungIndustry, EU, NuGo
Charité BerlinUlf GöbelStefan Bereswill
Biofunctionality, TUMDirk HallerIngrid SchmöllerSigrid KieslingThomas ClavelSusan ChangMicha HoffmannAnja MesslikTanja WernerEva RathGabriele HörmannspergerNatalie SteckEmanuel BergerMarie Anne von SchilldeStephan WagnerNico GebhardtBenjamin Tiemann
UNC, Chapel Hill, USABalfour R. SartorSandra C. Kim
DIfEMichael BlautGunnar Loh
Acknowledgements
Rowett InstituteDenise KellyCaroline Reiff
Top Related