Download View 2005 Investigator Meeting Presentation

Adaptive Immunity to Flagellin in IBD
Andrew Gewirtz, Ph.D.
Department of Pathology and Lab Medicine
Emory University, Atlanta GA
Background
E. Coli
Flagella
Flagellin monomers (About 70,000 per flagella)
Inestinal epithelial cell
?
NF-kB
Adaptive Immunity
Y
TLR5
Innate Immunity
Acute Inflammation
Cytokines
Neutrophil Influx
Hypothesis: Flagellin may be a target and
promoter of adaptive immunity in IBD
Methods
1.
Isolate Serum from IBD patients and control subjects
Fl
ag
A e lla
fl a te
ge d b
lla a
te cte
ba ria
ct
er
ia
2. Assess immunoreactivity to flagellins via western blotting or ELISA.
protein L-coated
Detect bound Ab with anti-IgA or IgG-HRP
flagellin-coated
LPS-coated
Detect product-specific Ab with anti-IgA or anti-IgG-HRP
14
CD #1
CD #3
Salmonella
Aflagellate Salmonella
E. coli
Aflagellate E. coli
CD #2
Salmonella
Aflagellate Salmonella
E. coli
Aflagellate E. coli
Salmonella
Aflagellate Salmonella
E. coli
Aflagellate E. coli
Normal #2
Salmonella
Aflagellate Salmonella
E. coli
Aflagellate E. coli
50
34
20
Normal #1
Salmonella
Aflagellate Salmonella
E. coli
Aflagellate E. coli
MW
205
97
Salmonella
Aflagellate Salmonella
E. coli
Aflagellate E. coli
Increased Immunoreactivity to
Bacterial Extracts in Crohn’s Disease
UC#1
Flagellin is a Target of Adaptive Immunity in Crohn’s Disease
LPS-specific IgG
Flagellin-specific IgG
1.25
Relative Total [IgG]
0.30
1.5
1.00
0.20
0.75
1.0
Optical density
0.50
0.10
0.5
0.25
0
Con
CD
UC
0
Con
CD
UC
0
LPS-specific IgA
Flagellin-specific IgA
Con
CD
UC
Relative total [IgA]
0.75
0.30
1.5
0.50
0.20
1.0
0.25
0.10
0.5
0
Con
CD
UC
0
Con
CD
UC
0
Con
CD
UC
Measuring immune responses to flagellin in mice
Inject flagellin (or controls) intraperitoneally
Innate Response
Cytokines at 90 min
Adaptive Response
Antibodies at 2 weeks
Mouse results/interpretation
Y
Flagellin
PC
IEC
Y
BC
TLR5
TNFα, IL-6
TC
APC
Y
Y
Carriage Of TLR5-stop Reduces Natural Acquisition
Of Adaptive Immunity To Flagellin
1.6
1.0
0.8
1.2
1.0
OD650
IgA
IgG
1.4
0.6
0.8
0.4
0.6
0.4
0.2
0.2
0.0
WT
Het
1:100
*
WT
Het
0.0
1:500
*: p<0.01
WT Het
1:100
WT Het
1:500
*
*
Reduced Rate of Crohn’s Disease In Persons Carrying TLR5-stop
(Jewish Cohort)
Number of subjects Het-TLR5-stop # (%)
Healthy Relatives
296
16 (5.4%)
Crohn’s Disease
215
2 (0.9%)
Ulcerative Colitis
185
11 (6.0%)
Ethnic Controls
416
27 (6.5%)
Conclusions
1. Common gram-negative flagellins (i.e. E. coli) are
antigenic targets of the elevated adaptive immune
response in Crohn’s disease.
2. Adaptive immunity to flagellin is regulated by
TLR5-mediated innate immunity.
3. Cytokines (e.g. IL-6 and TNFα) produced by nonhematopoietic cells, may link mucosal detection of
flagellin to adaptive immune response.
4. Adaptive immunity to flagellin may promote
development of Crohn’s Disease.
Acknowledgements
Emory University
Ifor Williams
Vijay Kumar
Dan Moore
Cathy Sanders
Cedars-Sinai/UCLA
Stephan Targan
University of Chicago
Judy Cho
Host Defense, Osaka, JP
Shizuo Akira