Regulation of macrophage inflammatory signaling pathways by AMP-activated protein kinase.

155 Pages·2016·3.11 MB·English

by  Yanfang Zhu

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Electronic Theses and Dissertations
5-2014
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Yanfang Zhu
University of Louisville
Follow this and additional works at: https://ir.library.louisville.edu/etd
Part of the Immunology and Infectious Disease Commons, and the Microbiology Commons
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Zhu, Yanfang, "Regulation of macrophage inflammatory signaling pathways by AMP-activated protein
kinase." (2014). Electronic Theses and Dissertations. Paper 1649.
https://doi.org/10.18297/etd/1649
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REGULATION OF MACROPHAGE INFLAMMATORY SIGNALING PATHWAYS
BY AMP-ACTIVATED PROTEIN KINASE

By

Yanfang “Peipei” Zhu
B.Sc. Northwest A & F University, 2007
M.S. University of Louisville, 2011

A Dissertation
Submitted to the Faculty of the
University of Louisville School of Medicine
in Partial Fulfillment of the Requirements
for the Degree of

Doctor of Philosophy

Department of Microbiology and Immunology
University of Louisville
Louisville, KY

May, 2014
Copyright 2014 by Yanfang “Peipei” Zhu

All rights reserved.

REGULATION OF MACROPHAGE INFLAMMATORY SIGNALING PATHWAYS
BY AMP-ACTIVATED PROTEIN KINASE

By

Yanfang “Peipei” Zhu
B.Sc. Northwest A & F University, 2007
M.S. University of Louisville, 2011

A Dissertation Approved on
April 25, 2014

by the following Dissertation Committee:

_________________________________
Jill Suttles

_________________________________
Thomas C. Mitchell

_________________________________
Robert A. Mitchell

_________________________________
Sanjay Srivastava

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DEDICATION

Dedicated to my beloved family and friends.

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ACKNOWLEDGMENTS

My highest regards go to my mentor, Dr. Jill Suttles. Six years of research
training in her lab has provided me one of the most important and valuable experiences in
my life, both professionally and personally. Dr. Suttles offered me the most wonderful
mentorship and the exciting opportunity to work on the scientific project that inspired me
in a life long academic interest. Without her guidance, support, encouragement, and
friendship, I could never have come this far on the way of pursuing the truth of nature.
My great appreciation also goes to my dissertation committee members, Dr. Thomas C.
Mitchell, Dr. Robert A. Mitchell, and Dr. Sanjay Srivastava, for their valuable comments
and advice and continuous care to the development of my project. I also want to express
my gratitude to the late members of my committee, Dr. Robert D. Stout and Dr. Michael
Martin. Bob was our beloved friend and insightful teacher whose lectures motivated me
to the macrophage plasticity research. Mike’s profound knowledge in cell signaling
made him always the good instructor to go to for suggestions. The scientific achievement
of this dissertation study is based on the contribution of every idea and support from all
these incredible scientists. It is my great honor and pleasure to work and have my Ph.D.
training with them.
I would like to thank all the nice people in Jill’s lab for their kind assistance and
support with all my experiments. I specially thank Lihua Zhang and her family, to
provide me the family-like support both in and out of lab work. I am grateful to all
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the nicest colleagues who have made the six years of training experiences cheerful and
have brought me courage through the downside of the road. They are Kim Z. Head,
Shunmugavadiv “Meena” Vanchinathan, Ashley Triplett, Kelly C. Caroll, Courtney J.
Mitchell, and Jonathan R. Brown. Thank you all for the scientific discussions and
technical support, for the laughter and compassionate hugs, and for the friendship that
will be remembered and appreciated life-long.
I also would like to thank my dearest family and friends. It is never enough to say
thank you to my parents Mingshan Zhu and Suyin Sun, for their best love and support
even with half of the world between us. You are the greatest blessing I can ever ask for. I
want to express my gratitude to my friends and family from the near and the far, Chi
Zhang, Pei-hsin Cheng, and my friends and big family in China. You all made me feel
beloved and encouraged during my studies in United States and made my life happy and
colorful.
Lastly, I want to thank Department of Microbiology and Immunology and the
Integrated Programs of Biomedical Sciences (IPIBS) program of University of Louisville
School of Medicine for the opportunity and financial support for the training of obtaining
my Ph.D. degree in immunology.
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ABSTRACT

REGULATION OF MACROPHAGE INFLAMMATORY SIGNALING PATHWAYS
BY AMP-ACTIVATED PROTEIN KINASE

Yanfang “Peipei” Zhu

April 25, 2014

AMP-activated protein kinase, AMPK, is a conserved serine/threonine kinase
with a critical function in the regulation of metabolic pathways in eukaryotic cells.
Recently, AMPK has been shown to play an additional role as a regulator of
inflammatory activity in leukocytes. Treatment of macrophages with chemical AMPK
activators, or forced expression of a constitutively active form of AMPK, results in
polarization to an antiinflammatory phenotype. Additionally, we reported previously that
stimulation of macrophages with antiinflammatory cytokines such as IL-10, IL-4 and
TGF-β results in rapid activation of AMPK, suggesting that AMPK contributes to the
suppressive function of these cytokines. In the current study we investigated the role of
AMPK in the regulation of macrophage antiatherogenic functions and demonstrated a
new mechanism for AMPK’s role in mediating IL-10-induced gene expression and
antiinflammatory effects. The expression of dominant negative (DN-) AMPKα1 in
macrophages resulted in a spontaneous obese phenotype in mice associated with fatty
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liver and heart enlargement. The expression of constitutively active (CA-) AMPKα1 in
macrophages resulted in decreased IL-6 production but increased expression of ABCA1
and ApoE. Compared to wild-type macrophages, AMPKα1-deficient macrophages failed
to express atheroprotective genes including ApoE, LXRα, and ABCA1 in response to IL-
10. Mechanistic studies revealed that IL-10-stimulated wild-type macrophages displayed
rapid activation of PI3K and its downstream target Akt, an effect that was not seen in
macrophages generated from AMPKα1-deficient mice. Treatment with the PI3K
inhibitor LY294002 blocked IL-10’s ability to induce Akt activation but not AMPK
activation, suggesting that IL-10-mediated activation of AMPK is independent of PI3K.
CA-AMPKα1 macrophages displayed elevated PI3K and CREB activation in response to
IL-10 compared to the empty vector transfected macrophages. IL-10 stimulation resulted
in increased mTORC1 activity, an Akt downstream target, an effect that was reduced in
AMPKα1-deficient mice. IL-10 induced phosphorylation of both Tyr705 and Ser727
residues of STAT3 in an AMPKα1-dependent manner, and these phosphorylation events
were blocked by inhibition of CaMKKβ, an upstream activator of AMPK, and by the
mTORC1 inhibitor rapamycin, respectively. The impaired STAT3 phosphorylation in
response to IL-10 observed in AMPKα1-deficient macrophages was accompanied by
reduced SOCS3 expression and an inadequacy of IL-10 to suppress LPS-induced
proinflammatory cytokine production. Overall, our data demonstrate that AMPKα1 is
required for IL-10 activation of the PI3K/Akt/mTORC1 and STAT3/SOCS3
antiinflammatory pathways regulating macrophage polarization, a mechanism with
broad-reaching applicability in immune homeostasis and in inflammation-associated
diseases such as atherosclerosis, autoimmune diseases, and obesity.
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