Mechanisms of Acute Inflammatory Control by Phagocytes by Aja M. Rieger A thesis submitted in ...

434 Pages·2014·6.09 MB·English

Mechanisms of Acute Inflammatory Control by Phagocytes

by

Aja M. Rieger

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in
Physiology, Cell and Developmental Biology

Department of Biological Sciences
University of Alberta

© Aja M. Rieger, 2014
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Abstract
Phagocytosis is an important, evolutionarily conserved mechanism integral to
immune defense and homeostasis. Phagocytosis is initiated by the interaction of
receptors on the surface of phagocytes with ligands on large particles, generally
over 1 μm. Receptor ligation results in actin polymerization, which subsequently
leads to particle internalization. The main objective of my thesis research was to
characterize functional phagocytic responses at inflammatory sites, where various
responses are involved in the clearance of pathogens, dying or senescent cells, and
in tissue repair/ wound healing. While this balance is well understood in mammals,
much less is known in lower vertebrates.
In accordance with mammals, I found that differentiation along the
macrophage pathway resulted in increases in phagocytic capacity, with mature
macrophage having the greatest capacity. Interestingly, activation resulted in
differential regulation of phagocytosis in monocytes and mature macrophages.
Within these subsets there was also differential regulation of phagolysosome fusion
and the production of reactive oxygen species (ROS). The activation of specific
phagocytic responses at distinct stages of differentiation suggests that these
responses may allow specialization of host immunity requirements within
specialized niches.
When phagocytic responses to inflammatory or homeostatic responses were
studied, I found that teleost fish displayed divergent responses following
phagocytosis, which is consistent with observations in mice. However, I found
significant differences between these species with regards to the level of
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responsiveness to zymosan and apoptotic bodies, the identity of infiltrating
leukocytes, their rate of infiltration, and the kinetics and strength of resulting
antimicrobial responses. The primary differences were noted in the neutrophilic
responses. Importantly, I found that activated murine, but not teleost, neutrophils
possess the capacity to internalize apoptotic cells, resulting in decreased neutrophil
ROS production. This may play an important part in the recently identified anti-
inflammatory activity that mammalian neutrophils display during the resolution
phase of inflammation.
Goldfish responses were partially regulated by soluble factors. However, no
changes were noted in canonical mammalian factors, leading to identification of a
novel role for a unique teleost receptor- soluble colony stimulating factor-1
receptor (sCSF-1R). Soluble CSF-1 receptor was originally described as a regulator
of macrophage proliferation. Soluble CSF-1R is highly upregulated following
interaction with apoptotic cells, resulting in reduced cellular infiltration,
phagocytosis, ROS production, expression of pro-inflammatory factors, and
downstream antimicrobial responses. The effects sCSF-1R mirrored several of
those induced by apoptotic cells, suggesting that sCSF-1R may be a central player
in the regulation of anti-inflammatory responses induced by apoptotic cells.
These observations were then applied to the study of an Aeromonas infection.
Aeromonas is a highly virulent fish pathogen with devastating effects on the fish
farming industry. It has been previously shown that increases in the production of
reactive intermediates by host immune cells cause significant cytotoxicity at the
infection site, but have limited protective effects against this catalase-producing
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bacterium. I found that Aeromonas veronii infection promotes systemic expression
of sCSF-1R. However, unlike other pathogen models, sCSF-1R was unable to
inhibit inflammation at the A. veronii challenge site and had no impact on the
production of ROS in vitro or in vivo. Importantly though, I found that the
increased systemic expression of sCSF-1R in A. veronii infected fish appears to
decrease proliferative activity among cells in the hematopoietic compartment,
which is further coupled to a decrease in CSF-1 expression in kidney hematopoietic
tissues.
Overall, the data presented here places the CSF-1 system and its regulator
sCSF-1R at the center of both the induction and regulation of inflammation in
teleost fish, with the effects of this system impacting the macrophage-lineage cells
as well as the neutrophilic and potentially lymphocytic cells.

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Preface
This thesis is an original work by Aja M. Rieger. The research project, of
which this thesis is a part, received research ethics approval from the University of
Alberta Research Ethics Board as part of an NSERC Discovery Grant entitled
"Comparative biology of phagocytic antimicrobial responses" awarded to Dr.
Daniel R. Barreda, held from 2008-2013 and 2013-2018:
1. Comparative biology of fish phagocytic antimicrobial responses;
Protocol # 706.
2. Comparative biology of phagocytic antimicrobial responses in mice;
Protocol # 760.
Some of the research conducted in this thesis was part of collaborations with
local and international groups, leading to publication. Details for each chapter are
outlined below:
Chapter 1: Portions of this chapter have been previously published in: Rieger
AM and Barreda DR. 2011. Antimicrobial mechanisms of fish leukocytes. Dev
Comp Immunol 35: 1238-45. And Rieger AM, Hanington PC, Belosevic M and
Barreda DR. 2014. Control of CSF-1 induced inflammation in teleost fish by a
soluble form of the CSF-1 receptor. Fish Shellfish Immunol pii: S1050-
4648(14)00105-3. For both manuscripts, I contributed to the design of the review,
manuscript composition, and figure design. P.C. Hanington contributed to a portion
of the manuscript composition. M. Belosevic contributed to manuscript editing.
D.R. Barreda was the supervisory author, and assisted in manuscripts composition
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and editing.
Chapter 3: A version of this chapter has been previously published in: Rieger
AM, Hall BE and Barreda DR. 2010. Macrophage activation differentially
modulates particle binding, phagocytosis and downstream antimicrobial
mechanisms. Dev Comp Immunol 34: 1144-59. I was responsible for the
experimental design, data collection and analysis, and manuscript composition.
B.E. Hall (Amnis Corporation, part of EMD Millipore) assisted in design of
analysis templates and contributed to manuscript editing. D.R. Barreda was the
supervisory author, and assisted in experimental design and manuscript
composition.
Chapter 4: A version of this chapter has been previously published in: Rieger
AM, Konowalchuk JD, Grayfer L, Katzenback BA, Havixbeck JJ, Kiemele MD,
Belosevic M, Barreda DR. 2012. Fish and mammalian phagocytes differentially
regulate pro-inflammatory and homeostatic responses in vivo. PLoS One 7: e47070.
I was responsible for experimental design, data collection and analysis, and
manuscript composition. J.D. Konowalchuk assisted with data collection from
murine experiments. L. Grayfer assisted with Q-PCR experimental design. B.A.
Katzenback assisted with neutrophil identification and analysis. J.J. Havixbeck
assisted with manuscript editing. M.D. Kiemele assisted in flow cytometry
staining. M. Belosevic and D.R. Barreda were supervisory authors, and assisted in
experimental design and manuscript editing.
Chapters 5 and 6: Portions of these chapters has been previously published
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in: Rieger AM, Konowalchuk JD, Havixbeck JJ, Robbins JS, Smith MK, Lund JM,
Barreda DR. 2013. A soluble form of the CSF-1 receptor contributes to the
inhibition of inflammation in a teleost fish. Dev Comp Immunol 39: 438-46. I was
responsible for experimental design, data collection and analysis, portions of the
manuscript composition, and manuscript editing. J.D. Konowalchuk and J.J.
Havixbeck were involved in data collection and manuscript editing. J.S. Robbins
was responsible for the generation of recombinant sCSF-1R. M.K. Smith assisted
in sample processing and manuscript editing. J.M. Lund provided preliminary data
that guided this line of research and assisted in manuscript editing. D.R. Barreda
was the supervisory author, and assisted in experimental design, manuscript
composition, and manuscript editing.
Chapter 8: A portion of this chapter has been previously published in: Rieger
AM, Hanington PC, Belosevic M and Barreda DR. 2014. Control of CSF-1
induced inflammation in teleost fish by a soluble form of the CSF-1 receptor. Fish
Shellfish Immunol pii: S1050-4648(14)00105-3. I contributed to the design of the
review, manuscript composition, and figure design. P.C. Hanington contributed to
a portion of the manuscript composition. M. Belosevic contributed to manuscript
editing. D.R. Barreda was the supervisory author, and assisted in manuscripts
composition and editing.
Appendix I: A version of this chapter has been previously published in:
Rieger AM, Hall BE, Luong Le T, Schang LM, Barreda DR. 2010. Conventional
apoptosis assays using propidium iodide generate a significant number of false
positives that prevent accurate assessment of cell death. J Immunol Methods 358:
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81-92. And Rieger AM, Nelson KL, Konowalchuk JD, Barreda DR. 2011.
Modified AnnexinV/Propidium Iodide apoptosis assay for accurate assessment of
cell death. J Vis Exp 50: pii: 2597. I was responsible for experimental design, data
collection and analysis, video performance, and manuscript composition for both
papers. B.E. Hall (Amnis Corporation, part of EMD Millipore) assisted in design of
analysis templates and contributed to manuscript editing. T. Luong Le preformed
all viral infections. L.M. Schang assisted in experimental design of viral work and
participated in manuscript editing. K.L. Nelson assisted in compiling figures for the
JoVE publication and assisted in manuscript editing. J.D. Konowalchuk assisted in
data collection, manuscript editing, and video performance. D.R. Barreda was the
supervisory author for both papers, and assisted in experimental design and
manuscript editing.

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Acknowledgements
This journey has been a fantastic and rewarding experience that would not
have been possible without the guidance, support, and encouragement of many
individuals. I appreciate everything you have done for me over the past 5 and a bit
years and would not be where I am today without you.
Firstly, I would like to thank my supervisor Dr. Dan Barreda. Dan, you have
provided me with excellent advice, suggestions, and opportunities, while allowing
me to grow as a scientist. Thank you for always being there to bounce ideas off of
and supporting (and funding) my research ideas, even when they sounded crazy at
the start. I have been spoiled in your lab and will be hard pressed to find another
lab with the same resources, mentorship, and atmosphere. It has truly been a
pleasure to work with you.
I would also like to thank my committee members, Dr. Mike (Miodrag)
Belosevic and Dr. Luis Schang. Your guidance, comments, and input have been
integral to the shaping of my research. I would also like to extend an extra thanks
to Mike for being like a co-supervisor, always being available for pop-ins from
across the hall or a beer at the Faculty Club. The science chats we have had
inspired me to be creative in my research and your curiosity and love of science is
truly infectious. I would also like to thank my defense and candidacy examination
members: Dr. Lisa Cameron, Dr. Christine Szymanski, Dr. Ben Willing, and Dr.
Jesús Lamas for thought-provoking and interesting discussions and challenging
questions. Finally, Dr. Allen Shostak, thank you for providing highly valuable
statistical advice.
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To my lab mates throughout my degree: you kept it real! My Ph.D.
experience would not have been nearly as much fun without having you at my side.
A special thanks to Moira Kiemele, Matthew Smith, Jeffrey Konowalchuk, Jeffrey
Havixbeck, and Mike Wong, as well as to my colleagues from the center wing
gang- you’ve all been a pleasure to work closely with, talk science with, and for
being fantastic friends in and out of the lab. Thank you for making Bio Sci a
magical place.
I have also had the opportunity to collaborate with a number of scientists
throughout the course of my degree. Dr. J. Oriol Sunyer, Dr. David Parra, and Dr.
Fumio Takizawa (UPenn); Dr. Miki Nakao and Takahiro Nagasawa (Kyushu
University); Dr. John Chang and Joshua Pemberton (U of A); Dr. A. Dean Befus
and Dr. Tae Chul Moon (U of A); Dr. Mike Belosevic and Belosevic lab members
(Dr. Leon Grayfer, Dr. Barbara Katzenback, Dr. Fumihiko Katakura; U of A); Dr.
Luis Schang (U of A), you have all provided me with the opportunity to broaden
my research and understanding of other research areas.
Finally, to my family and friends, especially my parents, thank you for your
undying support and encouragement, for keeping me sane and levelheaded, and for
helping wherever possible. I’d like to give a special thank you to Keith Heslinga-
your engineering logic helped me to think outside of the box and inspired some
truly imaginative ideas and solutions throughout my degree, and your constant
cheer-leading pushed me to be the best scientist I could be. To Azalia and Quince-
your smiles make even the longest, most frustrating days better.
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