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Immunomodulation of bone marrow-deriveddendritic cells during endotoxin- and bacterialsonicate-induced tolerance
Undergraduate Senior ThesisMicrobiology
 Tyler Cole
December 1
st
, 2009
Sponsor: John Y. Kao, MDUniversity of Michigan Medical School Department of Internal MedicineDivision of Gastroenterology Co-sponsor: Gary Huffnagle, PhDUniversity of Michigan
– 
Ann Arbor College of Literature, Science, and the Arts
 
 
 
2TABLE OF CONTENTS
ACKNOWLEGEMENTS 3
 
ABSTRACT 4
 
BACKGROUND AND INTRODUCTION 6
 
HYPOTHESIS 11
 
MATERIALS AND METHODS 12
 
RESULTS
17
 
FIGURE 
 
1
 
-
 
TNF-
Α EXPRESSION WITH VA
RYING LOW DOSES OF
LPS
TOLERIZATION AND SUBSEQUENT HIGHER DOSESTIMULATION
22
 
FIGURE 
 
2
 
-
 
TNF-
Α EXPRESSION BY
BM-DC
S DURING TOLERANCE TIME COURSE
23
 
FIGURE 
 
3
 
-
 
TNF-
Α EXPRESSI
ON DECREASES IN SUPERNATANT OF
DC
S TOLERIZED WITH
LPS,
 
H.
PYLORI
 
SS1
 SONICATE
,
OR
E.
COLI
SONICATE
24
 
FIGURE 
 
4
 
-
 
IL-12
P
70
EXPRESSION DECREASES IN SUPERNATANT OF
DC
S TOLERIZED WITH
LPS,
 
E.
COLI
SONICATE
,
 OR
H.
PYLORI
SS1
SONICATE
25
 
FIGURE 
 
5
 
-
 
IL-10
M
RNA
AND SECRETED PROTEIN EXPRESSION IN
DC
S TOLERIZED WITH
LPS
OR
H.
PYLORI
SS1
 SONICATE
26
 
FIGURE 
 
6
 
-
 
IL-10
SECRETED PROTEIN EXPRESSION IN
DC
S TOLERIZED WITH
LPS,
 
H.
PYLORI
 
SS1
SONICATE
,
OR
E.
 COLI
SONICATE
27
 
FIGURE 
 
7 
 
-
 
IL-6
EXPRESSION IN SUPERNATANT OF
DC
S TOLERIZED WITH
LPS,
 
H.
PYLORI
 
SS1
SONICATE
,
OR
E.
COLI
 SONICATE
28
 
FIGURE 
 
8
 
-
 
TNF-
Α EXPRESSION IN SUPE
RNATANT OF
DC
S TOLERIZED WITH
LPS
AND
H.
PYLORI
 
SS1
SONICATEWITH OR WITHOUT
IL-10
NEUTRALIZING ANTIBODY
29
 
FIGURE 
 
9
 
-
 
IRAK-M
M
RNA
AND PROTEIN EXPRESSION IN
DC
S TOLERIZED WITH
LPS
30
 
FIGURE 
 
10
 
-
 
TNF-
Α EXPRESSION IN SUPE
RNATANT OF WILD TYPE VS
IRAK-M
 
-/-
 
DC
S TOLERIZED WITH
LPS,
 
H.
 PYLORI
 
SS1
SONICATE
,
OR
E.
COLI
SONICATE
31
 
FIGURE 
 
11
 
-
 
IL-10
EXPRESSION IN SUPERNATANT OF WILD TYPE VS
IRAK-M
 
-/-
 
DC
S TOLERIZED WITH
LPS,
 
H.
 PYLORI
SS1
SONICATE
,
OR
 
E.
COLI
SONICATE
32
 
DISCUSSION 33
 
CONCLUSION 41
 
FUTURE DIRECTIONS 42
 
ABBREVIATIONS 43
 
REFERENCES 44
 
 
 
3
ACKNOWLEGEMENTS
 
Countless thanks must be given to John Y. Kao, MD for so generously allowing me towork in his lab, helping to develop a feasible project, and giving direction every step of the way.Without his advice and encouragement, this project would not have been possible. I could nothave asked for a more understanding and supportive mentor for this project. His lessons inexperiment design and valuing every experiment (whether it turned out well or not) wereinestimable. Thanks are due to Dr. Gary Huffnagle for initially putting Dr. Kao and myself incontact, volunteering to serve as a co-sponsor for this thesis, and providing evaluation. Dr.Osterholzer volunteered after a last minute request for an additional reader for the thesis, andfor that much gratitude is in order.Many thanks are due to Min Zhang for her invaluable support in interpreting data andteaching me essentially all the techniques used in this project, not to mention all the times shehelped with cell culture and analysis when I had to get to class. Thanks to Jay Luther, MD forcareer advice and plenty of help with experiments. Tomomi Takeuchi and Stephanie Owyangwere fantastic while helping with ELISA, and I owe them thanks. Andrea Heldsinger wasincredibly kind in teaching me Western blot, and Haiyan Liu filled in any gaps that were missing,and I owe them both thanks.Thank you to UROP for providing fellowship funding for research this summer.
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