THE EFFECTS OF RUBULAVIRUS INFECTION ON MACROPHAGE FUNCTION

BY

CAITLIN MATTOS BRIGGS

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Microbiology and Immunology

August 2011

Winston-Salem, North Carolina

Approved By:

Griffith D. Parks, Ph.D., Advisor

Examining Committee:

Gregory S. Shelness, Ph.D., Chair

Martha A. Alexander-Miller, Ph.D.

Rajendar K. Deora, Ph.D.

David A. Ornelles, Ph.D.

ii

ACKNOWLEDGEMENTS

To my best friend and husband John, I cannot say thank you enough. You have supported me through graduate school each and every day and I wouldn‟t be here without you. From listening to practice talks, to proof-reading papers and abstracts, and helping me find my way through the good and bad times that come with research, you have always been there. I am so lucky to be married to you. Thank you for helping me reach this goal. Next up is parenthood! After graduate school, this should be easy, right? To my wonderful parents. Thank you for teaching me from a very young age that I could be whatever I wanted when I grew up. Your constant love, encouragement, and guidance has made me the person I am today. Dad, thanks for always pushing me to do my best and never letting me give up on myself. Mom, thanks for always being there to listen on my bad days and helping me find my way. To my sister, thanks for making me laugh on the bad days. To my graduate mentor, Griff. I am so thankful I joined your lab. You have been a fantastic teacher. You have guided me over the rough patches of research, and allowed me to pursue my own ideas and develop as a scientist. Thank you for all of the opportunities you have given me, and for the encouragement to always be better. Thank you to my wonderful lab mates, Ellen, John, Maria, Patrick, Jerry, and Mary Jo. I could not have asked for a more wonderful group of people to work with! Mary, thank you for all of the conversations, scientific and personal. John, thank you for all of the advice and guidance you have given me. I‟ve enjoyed sharing a bench with you, and will miss your stories! Maria and Patrick, I‟ve missed you both. Thank you for your patience in training me during my first year. Ellen, thank you for all of your help with bench work, and for managing to keep our lab organized. To my wonderful friends and fellow students,I am so thankful to have met you all and am forever grateful for our friendship. Rick, Ro, and Amity, I know I couldn‟t have survived here without you guys. Thanks for all the fun, and for the support. Nikki, Amy, Latoya, Katie, Anne, Cheraton and Ashley, thank you for being my partners in crime! You‟ve made this grad school experience a fun one. All of the trips, dinners, and nights out will never be forgotten.

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TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS………………………………………………………………ii LIST OF FIGURES…………………………………………………………………..... iv ABBREVIATIONS………………………………………………………………………vi ABSTRACT………………………………………………………………………………x CHAPTER I. INTRODUCTION………………………………………………………..1 II. MATERIALS AND METHODS…………………………………….....25

III. MUMPS VIRUS INFECTION INHIBITS MIGRATION AND PHAGOCYTOSIS OF MACROPHAGES IN A TNF-α-DEPENDENT MANNER…………………………………………………………...…..30

IV. ACTIVATION OF MACROPHAGES BY BACTERIAL

COMPONENTS RELIEVES THE RESTRICTION ON REPLICATION OF AN INTERFERON-INDUCING PARAINFLUENZA VIRUS 5 (PIV5) P/V MUTANT………………………………………………………………..46

V. DISCUSSION…………………………………………………………..63

REFERENCES CURRICULUM VITAE COPYRIGHT INFORMATION

iv

LIST OF FIGURES

CHAPTER I

FIGURES PAGE

1. Macrophage origin and differentiation 4

2. Macrophage activation drives effector cel function. 7

3. Macrophage migration occurs following chemokine 10 binding to receptor.

4. Schematic of PIV5 mutants used in this study. 21

CHAPTER III

5. PIV5 productively infects macrophages and inhibits 32 migration.

6. MuV productively infects macrophages with little effect 34 on macrophage viability.

7. MuV infection inhibits migration towards a variety of 37 chemoattractants. 8. Inhibition of macrophage migration is exacerbated by 38 viral load and dependent upon viral replication. 9. Inhibition of macrophage migration during MuV infection 41 is caused by a soluble factor. 10. Neutralization of TNF-α is sufficient to restore migration 43 in MuV infected macrophages. 11. TNF-α is sufficient to inhibit migration in uninfected 45 macrophages.

v

CHAPTER IV

12. The P/V mutant is restricted for replication in primary 48 human macrophages. 13. IFN production by MDM contributes towards the 49 restriction of the P/V mutant. 14. Activation of primary human macrophages following 51 treatment with heat killed bacteria. 15. The P/V mutant is not restricted for replication in 54

primary MDM activated by Gram positive bacteria. 16. Alternatively activated MDM are not susceptible 55 to infection with the P/V mutant. 17. Monocytes display enhanced susceptibility to P/V 56 mutant infection following HKBac stimulation. 18. The increased susceptibility of activated MDM 58 to P/V mutant infection is transient. 19. MDM activated with HKBac maintain IFN signaling 61 but are deficient in IFN induction. 20. IRAK-M is higher in P/V mutant infected MDM activated 62 by HKBac.

CHAPTER V

21. A model for TNF-α-induced inhibition of macrophage migration. 73 22. A model for HKBac enhanced susceptibility to P/V mutant 81 infection in macrophages.

vi

ABBREVIATIONS

AP- activator protein APC- antigen presenting cell BCA- bicinchoninic acid BHI- brain-heart infusion CCL- C-C motif ligand CCR- C-C chemokine receptor CD- cluster of differentiation cDNA- complimentary deoxyribonucleic acid CNS- central nervous system CPE- cytopathic effects CPI- canine parainfluenza virus DAPI- 4',6-diamidino-2-phenylindole DC- dendritic cell ELISA- enzyme linked immunosorbent assay ERK- extracellular-signal regulated kinase F- fusion protein FACS- fluorescence activated cell sorting FBS- fetal bovine serum GFP- green fluorescence protein GMCSF- granulocyte macrophage colony stimulating factor GPCR- G-protein coupled receptor HCMV- human cytomegalovirus

vii

HEPES- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV- human immunodeficiency virus HKBA- heat-killed Bacillus anthracis HKBac- heat-killed bacteria HKLM- heat-killed Listeria monocytogenes HKGAS- heat-killed Streptococcus pyogenes HN- hemagglutinin and neuraminidase protein h pi- hours post infection HSC- haematopoietic stem cell IFN- interferon IL- interleukin IRAK- interleukin-1 associated receptor kinase IRF- interferon regulatory factor ISGF- interferon-stimulated gene factor ISRE- interferon-stimulated response element L- large protein LPS- lipopolysaccharide M- matrix protein MAPK- mitogen-activated protein kinase MCP- macrophage chemoattractant protein MCSF- macrophage colony stimulating factor mda- melanoma differentiation associated gene MDBK- Madin-Darby bovine kidney cells

viii

MDM- monocyte derived macrophage MFI- mean fluorescence intensity MIF- macrophage inhibitory factor MKP- MAPK phosphatase moi- multiplicity of infection MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MuV- mumps virus NEAA- non-essential amino acids NDV- Newcastle disease virus NFκβ- nuclear factor kappa β NK- natural killer NOD- nucleotide-binding oligomerization domain NP- nucleocapsid protein P- phosphoprotein PAGE- polyacrylamide gel electrophoresis PBMC- peripheral blood mononuclear cells PBS- phosphate buffered saline pDC- plasmacytoid dendritic cell PI3K- phosphoinositide-3 kinase PIV- parainfluenza virus PRR- pattern recognition receptor RANTES- regulated upon activation, normal T-cell expressed, and secreted RIG-I- retinoic acid-inducible gene 1

ix

RNA- ribonucleic acid RSV- respiratory syncytial virus SDS- sodium dodecyl sulfate SH- small hydrophobic protein SOCS- suppressor of cytokine signaling STAT- signal transducers and activators of transcription SV- simian virus TLR- toll like receptor TNF- tumor necrosis factor TOLLIP- toll interacting protein UV- ultraviolet VEGF- vascular endothelial growth factor VSV- vesicular stomatitis virus WT- wild-type

x

ABSTRACT

Caitlin Mattos Briggs

EFFECTS OF RUBULAVIRUS INFECTION ON MACROPHAGE FUNCTION

Dissertation under the direction of Griffith D. Parks, Ph.D.,

Professor and Chair, Department of Microbiology and Immunology

During viral infection, many different host cells can be targeted for

infection. While the interactions of viruses with all host cells are important, more

knowledge of how viruses interact with host immune cells is needed. The

presence of innate immune cells is critical in determining the outcome of viral

infection for the host. One important cell type for innate immune system is the

macrophage. In this thesis I have investigated the interactions of two

Rubulaviruses with macrophages: PIV5, which is a prototype Paramyxovirus and

Mumps Virus, which is the causative agent of mumps in humans.

PIV5 infection of macrophages results in a significant decrease in in vitro

chemotaxis. Similar inhibition of macrophage migration in vitro was seen

following Mumps Virus (MuV) infection. This inhibition in migration following MuV

infection was found to be independent of chemoattractant used and dependent

upon moi. Cytokine analysis revealed that TNF-α was produced following MuV

infection, and that this TNF-α was both necessary and sufficient to inhibit

macrophage migration.

xi

Our laboratory has developed a recombinant variant of PIV5 for potential

development as a viral vaccine vector and oncolytic therapy. This P/V mutant

virus encodes six amino acid substitutions in the shared P/V gene region which

converts WT PIV5 to a mutant that induces IFN and has lost the ability to block

IFN signaling. Infection of monocyte-derived macrophages (MDM) with the P/V

mutant resulted in restricted viral replication and the production of IFN-β, which

was at least partially responsible towards the restriction in replication. Activation

of macrophages with bacterial components enhanced replication of the P/V

mutant in MDM in a transient manner. While IFN-β signaling was found to be

intact in MDM activated by bacterial components, interestingly these activated

macrophages were found to be deficient in IFN-β induction upon subsequent P/V

mutant infection. The activation of MDM by bacterial components also resulted in

increased levels of the negative regulator IRAK-M, which has been shown to

block IFN induction pathways.

Taken together, the work described in this thesis indentifies two unique

mechanisms by which Rubulaviruses exploit host cell responses to enhance their

own ability to replicate and spread. These results address a gap in our

knowledge of how normal host innate immune responses are able to impact viral

infection.

1

CHAPTER I

INTRODUCTION

The ability of a host to mount an innate immune response to pathogens is

crucial to determining the outcome of infection. Cells of the host immune system

have evolved to detect invading viral and bacterial pathogens and to limit the

spread and scope of an infection. Likewise, viral and bacterial pathogens have

evolved their own means for subverting this detection and clearance. The

outcome of infection is often determined by which of these two mechanisms is

able to act the fastest and most effectively: the host immune cells, to clear the

infection or the pathogen, to replicate and spread. Often, a delayed response by

host immunity results in disease, which it is later able to overcome. It is of

increasing importance that we understand the mechanisms of how viral infection

can alter functions of immune cells, and protective immune responses can

sometimes work against the host instead of for its benefit, resulting in enhanced

viral and bacterial infection.

The overarching goal of this work is to understand the interactions of

Rubulaviruses with macrophages, and how viral infection affects macrophage

function. Rubulaviruses have evolved mechanisms to avoid the induction of

innate immune response. In the event that the virus is unable to avoid induction

of innate immunity, it may be able to take advantage of normal host cell

responses to benefit its own replication and spread. The mechanisms by which

Rubulaviruses take advantage of these normal macrophage responses to benefit

2

their replication is not well understood. Experimental work in my thesis defines

several unique mechanisms that viruses have evolved to either limit immune cell

function, or exploit immune cell responses to benefit their own replication. This

includes the induction of cytokines during viral infection of cells which limit the

ability of the host cell to migrate, as well as the ability to take advantage of an

immune cells activated state to propagate new virus. My work will be

summarized in two models: one where the inhibition of macrophage migration

during viral infection is due to enhanced cytokine production during viral infection,

and one where the increased susceptibility of macrophages to infection with P/V-

CPI- is due to normal regulatory control of host cell responses by the

macrophages following exposure to bacterial components. Both of these

mechanisms are examples of normal macrophage responses to pathogens that

are exploited by Rubulaviruses for their own benefit.

Macrophage origin and development. Macrophages are important antigen

presenting cells (APC) which, like dendritic cells, are responsible for the

regulation of innate and adaptive immune response during microbial infections

(5, 95, 32, 93, 48). Macrophages are present in virtually all tissues, and act

locally, by destroying pathogens and maintaining inflammatory responses at the

site of infection (65, 57). The main functions of macrophages are to recognize,

phagocytose and destroy infectious agents, and then present microbe-specific

peptides to lympocytes (57).

3

Macrophages are differentiated from circulating peripheral blood

mononuclear cells (PBMCs) which have been found to migrate into tissues either

in a steady state, or during inflammation (36). The origination of these PBMCs is

from a common myeloid progenitor cell found in the bone marrow, called a

haematopoietic stem cell (HSC). During monocyte development, HSCs give rise

first to myeloid progenitor cells, then to monoblasts following exposure to

macrophage colony stimulating factor, then pro-monoblasts and finally

monocytes (65). Once they have achieved monocyte status, they are released

from the bone marrow and into the blood stream. These monocytes are then

able to migrate into various tissues from the bloodstream to become long lived

resident macrophages which are tissue-specific such as for the bone

(osteoclasts), lung (alveolar macrophages), central nervous system (microglial

cells), connective tissue (histocytes), gastrointestinal tract, liver (Kupffer cells),

and spleen (36).

In humans, most monocytes appear to belong to one of two subtypes;

CD14hiCD16- which are defined as „classical‟ monocytes, or CD14-CD16+ cells

which are considered „non-classical‟ monocytes (36). Greater than 90% of

human monocytes express the classical markers (65). In in vitro culture,

classical monocytes can give rise to dendritic cells in the presence of granulocyte

macrophage colony stimulating factor (GMCSF) and interleukin-4 (IL-4), or

macrophages in the presence of GMCSF or macrophage colony stimulating

factor (MCSF) alone (39, 57, 36). Macrophages derived from

4

Figure 1. Macrophage Origin and Differentiation.

Figure 1. During development, bone marrow derived monocytes are released into the blood. From there they are thought to migrate into tissues to differentiate into different resident immune cells. In in vitro culture monocytes are able to be driven down the dendritic cell or macrophage lineage depending on cytokines used to differentiate the cells.

CD14+ monocyte

Macrophage

Dendritic cell

Adult tissueAdult peripheral blood

5

the simulation of monocytes with MCSF are considered to be naïve and

susceptible to activation (39). There are still questions remaining about the

heterogeneity of the monocyte population, primarily whether discrete monocyte

populations give rise to specific tissue macrophage populations (65).

Macrophage activation. Once monocytes have been driven down the

macrophage lineage, they are able to respond to endogenous stimuli, which

result in a dramatic, yet transient effect on the physiology of macrophages (65).

The type of stimuli received and the response of a naïve macrophage to these

stimuli can result in different populations of activated macrophages. While some

literature suggests that macrophage activation exists as a spectrum rather than a

linear designation (65, 39), there are generally two accepted subclasses of

activated macrophages; M1 and M2. M1 activated macrophages are typically

considered „classically activated‟, whereas M2 macrophages are thought to be

„alternatively activated‟ and function primarily in wound repair and healing (65,

39, 13, 35). For the purposes of this work, I will be focusing on the development

and characteristics of classical or M1 macrophages.

Originally, classically activated macrophages were characterized as

macrophages which had been activated through a combination of two signals;

interferon gamma (IFN-γ) and tumor-necrosis factor (TNF). It was observed that

these macrophages had heightened microbicidal activity and secreted high levels

of pro-inflammatory cytokines and their mediators (65, 62, 73). The origin of the

TNF-α and IFN-γ needed to produce M1 macrophages was originally shown to

6

be produced by other immune cells such as natural killer (NK) cells during the

course of infection (24). The production of IFN-γ by NK cells during infection is

usually transient, and so adaptive immune responses are required to enhance

and continue the response of M1 activated macrophages to infection (65). While

the activation of M1 macrophages was originally thought to require two signals

produced by adjacent immune cells, it has since been shown that macrophages

can achieve and M1 activation state through pattern recognition receptor (PRR)

signaling and the production of additional cytokines such as IFN-β through

signals that they receive during viral or bacterial infection, without the presence

of additional immune cells (65, 24).

Macrophage migration. Naïve and activated macrophages perform a variety of

functions to help overcome an infection; however, first they must be recruited to

the site of infection through a process known as migration or chemotaxis. At the

site of an infection, immune cells release a variety of cytokines and chemokines,

which can be constitutively produced by the cells, or induced upon stimulation.

Some of these chemokines serve to recruit macrophages to the site of infection

to help fight the pathogen. It has been proposed that this rather non-

7

Figure 2. Macrophage activation drives effector cell function.

Figure 2. Macrophages are able to be activated in a variety of ways, and the stimuli they encounter for activation is important in determining macrophage function. Classically activated macrophages arise from macrophages stimulated via pathogen encounter, or by exposure to cytokines from other innate cells such as NK cells. Classically activated macrophages are highly microbicidal and have enhanced phagocytic abilities compared to naïve macrophages.

IFN-γ

TNF-α

Y

TLR ligand

Naïve tissue macrophage

Classically activated

“M1”

IL-10

Prostaglandins

Apoptotic cells

Alternatively activated

“M2”

8

specific immune response is able to protect a host from at least 98% of the

pathogens it encounters (50). The chemokines are a group of at least 47 related

proteins, making them one of the largest families of cytokines known (82). These

chemokines are able to bind to G protein-coupled receptors or other surface

receptors such as tyrosine kinase receptors on the surface of macrophages and

are highly regulated (33, 105). As a result of the engagement of these receptors,

macrophages are stimulated to rearrange their cytoskeleton, to polarize, and

then to migrate towards the chemoattractant, which commonly thought of as a

gradient (80). The forward movement of macrophages after encountering a

chemoattractant can be divided into several steps. First, filopodia and

lamellipodia protrude at the leading front of the cell. Next, the protruding

filopodia and lamellipodia adhere to the substratum via focal adhesions. These

focal adhesions are large protein complexes through which the cytoskeleton of a

cell connects to the extracellular matrix. After adhesion has taken place, the

cytoplasmic actomyosin begins to contract, which leads to the release of the tail

end of the cell from the surface (50).

The chemokines studied in this thesis are MCSF, macrophage

chemoattractant protein 1 (MCP-1 or CCL2), Regulated upon Activation, Normal

T-cell Expressed, and Secreted protein (RANTES or CCL5), and Vascular

Endothelial Growth Factor (VEGF). MCSF is constitutively expressed in vitro by

several cell types such as endothelial cells, macrophages, osteoblasts, and

fibroblasts and exists in several isoforms. It is able to recruit macrophages to

sites of inflammation and activate them (39). MCSF is expressed at higher levels

9

at sites of inflammation and autoimmunity, and its elevated expression is found to

correlate with a variety of autoimmune disease states, including arthritis,

nephritis, and chronic lung inflammation (39).

MCP-1 is a potent chemotactic factor for monocytes and macrophages,

and is produced constitutively by a variety of cell types such as endothelial cells,

fibroblasts, monocytes, and microglial cells, although elevated production is

noted primarily from monocytes and macrophages after exposure of cells to

oxidative stress, cytokines, or growth factors (26). While MCP-1 induces

chemotaxis primarily in macrophages, it is also able to induce migration and

infiltration of monocytes, memory T lymphocytes, and NK cells (26). Inhibition of

MCP-1 is a common treatment for various diseases, such as multiple sclerosis,

arthritis and insulin-resistant diabetes (26).

RANTES is a chemokine produced by epithelial cells, lymphocytes, and

platelets in response to infection with pathogens such as respiratory syncytial

virus (RSV) and influenza virus, and is a potent chemoattractant for monocytes

and macrophages (60). While the production of RANTES during infection and

inflammation is thought to be important for determining pathogen clearance, the

exact mechanisms by which RANTES influences innate immune responses is

unclear (60).

VEGF is produced by tumor cells during growth and is specific for

endothelial cells (6). VEGF is a potent chemoattractant for monocytes and

macrophages and binds with high affinity to receptors on the

10

Figure 3. Macrophage migration occurs following chemokine binding to

receptors.

Figure 3. Macrophage migration is stimulated by chemokine binding to two different types of receptors; seven transmembrane domain receptors and tyrosine phosphotase receptors which detect M-CSF and MCP-1. Signaling pathways between these two receptors differ, but eventually converge upon activation of NFκB, leading to macrophage migration.

surface of these cells, and this binding is thought to not only lead to the migration

of macrophages and monocytes but also their activation (6).

While the ability of a macrophage undergo chemotaxis and migrate

towards the site of an infection is undoubtedly important, it is just as important to

Figure 3. Macrophage migration is stimulated by chemokine binding to two different types of receptors: seven transmembrane domain receptors and tyrosine phosphatase receptors. Signaling pathways between these two receptors differ, but eventually converge upon activation of NFκB, leading to macrophage migration.

chemoattractants

cytoplasm

MAP3K

MAP2K

MAPK

nucleus

NF κ B

NF κ B MIGRATION

Cell membrane

11

surface of these cells, and this binding is thought to not only lead to the migration

of macrophages and monocytes but also their activation (6).

While the ability of a macrophage to undergo chemotaxis and migrate

towards the site of infection is undoubtedly important, it is just as important to be

able to stop this process, to arrest cell migration and retain the macrophages at

the infection. However, a fine regulation of macrophage trafficking is needed to

avoid chronic inflammation, impaired tissue healing, and recurrent infection in a

host (33). Therefore, it is important that macrophages are readily able to move to

the site of an infection, be retained in those tissues to combat a pathogen, and

then egress out of the tissues to avoid damaging the host. Using a mechanism

that I will detail later in this thesis, MuV is able to prevent the migration of both

infected and non-infected macrophages through the production of a soluble

factor. I speculate that this inhibition of chemotaxis would limit the egress of

macrophages from the site of infection, resulting in a delay in adaptive immune

response, and contributing to swelling and tissue damage at the site of infection.

Upregulation of cytokines and co-receptors by macrophages. Activated

macrophages are able to release pro-inflammatory cytokines and type I IFN after

encountering pathogens. The premise behind this ability is multifaceted; the

production of cytokines may impair viral or bacterial growth, and these cytokines

are also able to recruit additional immune cells to the site of infection. Classically

activated macrophages produce specific cytokines and chemokines in response

12

to infection, and additionally upregulate a variety of surface markers which can

serve to activate adaptive immune responses (65).

For the purposes of this study, we evaluated the production of the

cytokines interleukin-6 (IL-6), TNF-α, RANTES, and macrophage inhibitory factor

(MIF). IL-6 is involved with the activation of acute phase response which can

play an important role in the early neutralization of pathogens (89). IL-6 is also

needed for activation of adaptive immune responses through inducing T cell

proliferation and enhancing B cell immunoglobulin expression (52). IL-6 has

been shown to be produced by activated macrophages in response to infection

(65). TNF-α is also produced during inflammation and infection, and like IL-6 is

involved in the acute phase response. TNF-α is produced by various cells of the

innate immune system and can also be produced by and activate macrophages

(65). TNF-α is important for the organization of humoral immune responses

especially those involved in B cell and dendritic cell (DC) follicular development

(76). The function of RANTES was described above, but additionally it is also

produced by macrophages during viral infection, and serves to provide

antiapoptotic signals to enhance survival (99).

MIF is an important cytokine discovered almost forty years ago during

studies of the delayed hypersensitivity reaction, and was one of the first

cytokines identified (12). While T cells were once thought to be the main source

of MIF, it was subsequently discovered that monocytes, macrophages, dendritic

cells, B cells, and eosinophils can all produce MIF; indeed MIF is produced

constitutively by macrophages at low levels (17). MIF is thought to bind to

13

receptor CD74 on macrophages, and is thought to activate extracellular-signal

regulated kinase (ERK) 1 and 2. MIF production is elevated when cells

encounter pathogens or pro-inflammatory cytokines, and this production is

thought to increase the ability of cells such as macrophages to phagocytose

pathogens and become activated upon pathogen encounter (17). However,

studies have also shown that the production of MIF by macrophages is sufficient

to inhibit the migration of macrophages to chemoattractants such as MIP-1 and

MCP-1. It has also been shown that some viruses such as human

cytomegalovirus (HCMV) induce MIF during infection in macrophages resulting in

paralyzed cells (33). This demonstrates that while MIF is an important cytokine

for innate immune response, it may also have undesirable effects on the ability of

macrophages to respond to subsequent infections.

In addition to producing cytokines to activate innate and adaptive immune

responses, activated macrophages will also upregulate surface markers in

response to viral infection. These receptors are able to simultaneously regulate

numerous pro-inflammatory responses and are important for innate immunity

(70). CD80 and CD86 are one class of co-stimulatory receptors, which are

activated via CD28 (72, 88, 38). While CD80 and CD86 are thought to have

overlapping functions, recent studies suggest that CD80 and CD86 have

differential regulation in the inflammatory response in vivo (44, 71). Activated

macrophages will upregulate CD80 and CD86 to enhance T cell stimulation and

activation of adaptive immune response (65).

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Type I IFN induction and signaling. My work also addresses the production of

type I IFNs, specifically IFN-β during virus infection. Like most nucleated cells,

macrophages are able to produce type I IFNs in response to pathogens (65).

IFN-β is secreted from macrophages and binds to its receptor to initiate signaling

in an autocrine or paracrine manner which leads to the induction of an antiviral

state within cells. Induction of IFN-β also contributes to the activation of the

adaptive immune response by activating immune cells such as NK cells and

dendritic cells (11). The levels of IFN-β and other pro-inflammatory cytokines

produced during viral infection play a significant role in determining the outcome

of viral infection.

The ability of a cell to enter an antiviral state is primarily dependent upon

the induction and signaling of type I IFNs. During type I IFN induction, viral

products are detected by a host cell by cytoplasmic host cell sensors, which

ultimately results in the activation of three latent cytoplasmic factors; nuclear

factor kappa β (NFkβ), interferon regulatory factor 3 (IRF-3), and activator protein

1 (AP-1). These three activated factors then translocate to the nucleus of the

cell, where they bind the IFN-β promoter and initiate the transcription of the IFN-β

gene. Once translation occurs, IFN-β is secreted from an infected cell.

During the IFN signaling phase, secreted type I IFN can act in an

autocrine or paracrine manner, by binding to the type I IFN receptor on the cell

surface. Binding of IFN-β to this receptor signals Jak and Tyk kinases to

phosphorylate and causes the dimerization of signal transducers and activators

of transcription (STAT) 1 and 2 (45). The STAT 1/2 heterodimer associates with

15

IRF-9, which forms the interferon-stimulated gene factor 3 (ISGF3). ISGF3 then

binds the interferon stimulated response element (ISRE) within the promoter of

target genes responsible for initiating an antiviral state within the host cell.

WT PIV5 is known as a poor inducer of pro-inflammatory cytokines,

including the induction of type I IFN (28, 55, 102, 103). This is due to the

cytoplasmic domain of the V protein which is able to block MDA-5 signaling, and

control viral replication. Additionally, the V protein is able to target STAT1 for

degradation during IFN signaling. This prevents the detection of WT PIV5 by the

host cell (102, 103). By contrast, the P/V mutant is a potent inducer of type I IFN,

despite having a functional V protein C-terminal domain (28, 102, 103). This is

due to the production of dsRNA during viral infection which is detected by the

cytoplasmic sensor RIG-I during infection (64). Additionally, the P/V mutant has

lost the ability to target STAT 1 for degradation, making it unable to block IFN

signaling (28, 102, 103).

Negative regulators of macrophage signaling. The ability of macrophages to

become activated and upregulate co-stimulatory molecules, and produce

cytokines, chemokines, and type I IFN in response to infection is crucial for the

host‟s ability to fight infection. However, it is also crucial that these responses be

tightly controlled to avoid damage to the host. For this reason, there are a variety

of host-derived mechanisms which have evolved to limit or reduce immune

responses. These mechanisms are termed negative regulators of signaling.

16

Negative regulators were first shown to play a role in dampening a

signaling response induced through toll-like receptor (TLR) signaling (59).

However, it has since been shown that negative regulators can be induced

through a variety of cellular signaling mechanisms, including nucleotide-binding

oligomerization domain containing 2 (NOD2) and retinoic acid-inducible gene 1

(RIG-I) (40, 84). More that ten different negative regulators of signaling have

been identified which are elicited in response to prolonged or strong stimuli of a

receptor by a pathogen (59). Several of these negative regulators of signaling

act intracellularly, such as suppressor of cytokine signaling (SOCS) 1,

phosphoinositide-3 kinase (PI3K), toll interacting protein (TOLLIP), A20, and IL-1

receptor-associated kinase-M (IRAK-M) (59). Perhaps the best characterized

intracellular negative regulator is SOCS-1. It has been shown that SOCS1 is

important for the suppression of cytokine signaling, and that mice deficient in

SOCS1 die shortly after birth due to multi-organ inflammation (91). SOCS1 has

been shown to directly target IRAK-1, inhibiting downstream NFκB activation

following LPS-induced signaling (59). For the purposes of my work, I will be

focusing on the role of IRAK-M in signaling inhibition.

The IRAK family of kinases is comprised of four members; IRAK-1, 2, 4,

and M (59). However, unlike the other members of the IRAK family, IRAK-M

expression is restricted solely to monocytes and macrophages (53,106). IRAK-M

lacks kinase activity and also negatively regulates pathogen induced signaling,

which is also in direct contrast to the other members of the family (53, 87). While

IRAK-M was originally thought to only be induced following TLR4 stimulation in

17

macrophages (59), subsequent research has shown that elevated IRAK-M

expression can be induced in macrophages through prolonged stimulation of

NOD2 by bacterial or viral stimulation (67, 83). Increased IRAK-M production is

thought to result in decreased pro-inflammatory cytokine and type I IFN

production during continued or subsequent stimulation of the cell by binding to

IRAK-1 and preventing downstream signaling (53, 59, 87,).

The Paramyxoviruses. The Paramyxoviridae are a family of enveloped, single-

stranded nonsegmented negative-sense RNA viruses. The genome of various

Paramyxoviruses ranges from 15-19 kilobases and can encode between 6-10

genes. Paramyxoviruses that are associated with a large number of diseases in

humans including measles virus, mumps virus, the human parainfluenza viruses,

respiratory syncytial virus, and the emerging Hendra and Nipah viruses. This

work focuses on the Rubulaviruses mumps virus (MuV) and parainfluenza virus

(PIV) 5, formerly called simian virus 5 (SV5).

PIV5 is a prototype Rubulavirus that has been studied as such for more

than fifty years. The genome of PIV5 is 15,246 bases and contains seven genes

which encode 8 proteins, arranged in order from 3‟-5‟; NP-P/V-M-F-SH-HN-L. A

schematic representation of the PIV5 genome is shown in Figure 4. The PIV5

virion is pleomorphic and composed of the negative-strand RNA genome

encapsulated in the viral helical nucleoprotein (NP). The matrix (M) protein is

found lining the inside of the lipid envelope. PIV5 contains two glycoproteins

hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein, which are

18

embedded within the lipid envelope of the virus and are exposed on the outer

surface of the virion. The cellular receptor for PIV5 is sialic acid and it is bound

by the HN protein during attachment of the virion to a host cell. The F protein

mediates the fusion of the viral envelope with the host cell plasma membrane, at

a neutral pH. The large (L) protein and the phosphoprotein (P) are also found

within the virion, and make up the viral RNA-dependent, RNA polymerase. The L

protein functions as the catalytic subunit of the polymerase, with the P protein

functioning as the anchor between the viral genome and the L protein.

The P/V gene is unique in the PIV5 genome because it encodes two

proteins, the P protein and the V protein. Two viral RNA transcripts are produced

from the P/V gene through a process called RNA editing, which occurs when the

viral polymerase “stutters” at a specific site in the P/V gene. This stuttering

results in the insertion of two nucleotides to the transcript, which effectively shifts

the translational open reading frame (97). During viral infection, faithful

transcription of the P/V gene results in synthesis of the V mRNA. However,

about 50% of the time the polymerase will slip and stutter, adding two non-

template G residues at a particular editing site within the RNA. This leads to a

shift in the open reading frame, which results in the production of the P protein

mRNA (97). As a result of this RNA editing, the P and V proteins share a 164

residue N-terminal domain, but have different and distinct C-terminal domains. A

PIV5 mutant used in this work, P/V-CPI-, contains substitutions in the shared N-

terminal region of the P/V gene, and its genome is depicted in Figure 4.

19

Wild-type (WT) PIV5 is able to establish highly productive infections with

minimal induction of host cell antiviral responses (22, 102). Our lab studies

naturally occurring variants of PIV5, which unlike WT PIV5 are potent activators

of host antiviral responses. Canine parainfluenza virus (CPI+) is closely related

to PIV5 and was originally isolated from a dog with a neurological disorder (9). A

naturally occurring variant of CPI+ was obtained through infection of a dog brain,

and this variant was termed CPI- due to its inability to degrade STAT1, and

prevent the induction and signaling of type I IFN (20). Genetic analysis revealed

that mutations to the shared N-terminal region of the viral P and V proteins were

responsible for these changes in CPI-. Using six amino acid substitutions

obtained from the P/V gene region of CPI-, our lab generated P/V-CPI- in the

backbone of WT PIV5 as shown in Figure 4. Like the parental CPI- virus, P/V-

CPI- is unable to target STAT1 for degradation, thus losing its ability to block type

I IFN signaling. P/V-CPI- has been shown to be a potent inducer of pro-

inflammatory cytokines and type I IFN, in direct contrast to WT PIV5 (103). P/V-

CPI- has been shown to rapidly induce caspase-dependent cytopathic effects

(CPE), also in direct contrast to WT PIV5 (28). This mutant virus is currently

being developed by our laboratory as a potential viral vaccine vector (3, 18). In

this thesis I used P/V-CPI- to examine enhanced viral replication in macrophages

following exposure to bacterial components.

MuV is also a member of the Paramyxoviridae, and is closely related to

PIV5, with high levels of homology of the viral proteins between the MuV and

PIV5. However, while PIV5 is not associated with any known disease in humans,

20

MuV is the causative agent of mumps, a viral infection of children and

adolescents characterized by swelling of the parotid glands (49). Additionally,

unlike PIV5, MuV infection is restricted to humans and has no known animal host

(49). In the era before vaccination arose, greater than 90% of adolescents 14-15

years old were seropositive for antibodies against MuV, indicating that the

majority of the population contracted the disease at some point during childhood.

MuV infection often resulted in secondary complications, including sterility in

males, aseptic meningitis, and deafness (58). While the introduction of a one-

dose MuV vaccination was successful in reducing the numbers of MuV cases

seen in recent years, there has been an increase in MuV outbreaks in vaccinated

populations, and an increase in unvaccinated populations, most likely due to the

decline in childhood vaccinations (49).

Bacterial and viral co-infections. Bacterial and viral co-infections are of

increasing concern in a clinical setting, and understanding the mechanism by

which infection with one organism predisposes a host to infection with a

subsequent pathogen is an important step in improving the treatment and

diagnostics of mixed pathogen infections. The most common model for mixed

infections involves influenza virus and Streptococcus pneumoniae. In this model,

initial infection with influenza virus renders a host susceptible to secondary

bacterial pneumonia infections caused by Streptococcus pneumoniae, and this

phenomenon is of concern during influenza pandemics (15, 92). Clinically,

21

Figure 4. Schematic representations of PIV5 and PIV5 mutants used in this

study.

Figure 4. The genome of PIV5 is depicted with the addition of the green fluorescence protein (GFP) gene between the HN and L genes. The P/V gene region has been enlarged to show the six amino acid substitutions inserted into the shared P-V gene region which result in P/V-CPI-. The WT PIV5 and CPI- sequences are shown below.

NP P/V M F HN L 5'GFP3'

N H 2

P / V R e g i o n

* * * V i r u s

PI V 5

C P I M i n u s

Y L

P

V T

H I I

L

P

S

F

1 5 7 1 0 2 5 0 3 3 3 2 2 6 * * *

22

secondary bacterial infections occur as the primary viral infection is being cleared

from the host by the immune system (15). Recent research has demonstrated

that immune responses elicited against the virus result in the decreased ability of

the host to fight secondary bacterial infections (94).

However, despite our understanding of how viral infection predisposes a

host to secondary bacterial infection, very little is known about the reverse

situation, in which a primary bacterial infection can enhance susceptibility of a

host to viral infection. Given that macrophages are an important cell type for

innate and adaptive immune responses (5, 65) and that activated macrophages

differ from naïve macrophages in their response to viral infection (3, 4, 96, 113)

my work focused on how macrophages activated by bacterial components would

differ in their response to a PIV5 mutant virus infection.

The ability of cells to change susceptibility to virus infection after exposure

to bacterial components has been reported previously for HIV infection of

macrophages (3, 113). Other groups have also shown that in an in vivo mouse

model of HIV-1 infection, the addition of heat killed Gram positive bacteria

enhanced HIV-1 protein production, and macrophages isolated from these mice

display reduced cytokine secretion and increased susceptibility to infection with

HIV-1 in vitro (113). Similarly, recent work from Nguyen et al. (2010) have shown

that RSV replication is enhanced when immature DC or primary epithelial cells

are treated with the TLR2 agonist Pam3CSK. Differences between the results I

obtained in this thesis and the work of others will be discussed later.

23

My work uses a panel of Gram positive bacteria to enhance susceptibility

to macrophages in this work, including Streptococcus pyogenes, Listeria

monocytogenes, and Bacillus anthracis. Briefly, Streptococcus pyogenes is the

causative agent of many important human diseases, ranging from mild illnesses

such as pharyngitis and impetigo to life threatening systemic infections such as

necrotizing fasciitis and toxic shock syndrome (81). Listeria monocytogenes is

an intracellular pathogen responsible for causing listeriosis and is one of the

most virulent food-borne pathogens in the United States (79). Bacillus anthracis

is the causative agent of anthrax, and is a pathogen of the respiratory tract, skin,

or digestive tract. It has been used as a biological weapon, and is most

commonly treated with antibiotics (75).

Summary of thesis work. The focus of this work was to gain understanding of

how viral infections such as MuV and PIV5 effect functions of macrophages such

as chemotaxis, and how naïve versus activated macrophages differ in their

responses to viral infection. Much of the literature is focused on identifying how

the innate immune system responds to viral infection; very little literature is

devoted to how immune functions may change when cells of the innate immune

system are infected. Likewise, there is a considerable bit of literature on how

viral activation of immune cells such as macrophages leads to enhanced

bacterial infection, but there is little known on how the bacterial activation of such

cells enhances susceptibility to subsequent viral infection.

24

My studies have shown that viral infection of naïve macrophages results in

an inability of these cells to migrate towards a variety of chemokines. The

retention of cells and their inability to migrate towards other chemoattractants

appears to be influenced by the amount of TNF-α secreted by cells during viral

infection. The addition of TNF-α alone was sufficient to inhibit macrophage

migration.

Additionally, studies were carried out to determine how macrophages

activated by bacterial components were able to respond to infection with a

previously restricted virus, P/V-CPI-. Whie naïve macrophages were resistant to

infection with P/V-CPI-, enhanced viral replication was seen in macrophages that

were activated by exposure to a variety of Gram positive bacteria. This

enhanced viral infection was attributed to a lack of antiviral responses in these

cells, most notably a deficiency in the production of IFN-β during subsequent viral

infection.

This work offers an important contribution to our understanding of how

viral infection may affect macrophages. It proposes novel mechanisms by which

the ability of the host to clear viral infection or defend itself against secondary

pathogens may be compromised during Rubulavirus infection or following

bacterial exposure.

25

CHAPTER II

MATERIALS AND METHODS

Cells, viruses and bacteria. Immature peripheral blood mononuclear cells

(PBMCs) were derived from whole human blood as described previously (3).

Briefly, PBMC were isolated from randomly selected donors by standard density

gradient centrifugation on Ficoll-Hypaque. CD14+ monocytes were isolated by

positive selection using CD14+ micro beads (Miltenyi-Biotec). Generally, the

isolated cells were found to be ≥ 95% CD14+ by flow cytometry. Enriched

CD14+ monocytes were cultured in RPMI media (Lonza) containing 10 ng/mL

hMCSF (Invitrogen) for six days supplemented with 10% FBS, and 1% each of L-

glutamine, penicillin/streptomycin, NEAA, 1M HEPES, and sodium pyruvate

(Lonza). After 3 days in culture, half of the media was replaced, and additional

hMCSF was added at the concentrations listed above. At day 6, ≥ 90% of cells

were CD64+, CD14+ (not shown).

WT PIV5 expressing green fluorescent protein (PIV5-GFP) was recovered

as described previously from a cDNA plasmid kindly provided by Robert Lamb

(Northwestern University) and Biao He (University of Georgia) (102). PIV5-GFP

stocks were grown in MDBK cells. rPIV5-P/V-CPI- GFP was recovered as

described previously (102). Virus stock for P/V-CPI- was generated in Vero cells

by low moi infection as described previously to prevent generation of defective

26

particles (34). Mumps Virus (Enders Strain, ATTC VR-1379) was grown in

MDBK cells.

Streptococcus pyogenes (MGAS5005 strain) was cultured as described

previously (81) in Todd-Hewitt broth (Becton-Dickinson) supplemented with 2%

yeast extract (Fisher Scientific). Bacillus anthracis (Sterne strain) was cultured

as described previously in brain-heart infusion broth (BHI; Becton-Dickinson)

(75). Haemophilus influenzae was grown on BHI plates by streaking at 37°C. .

Bacteria were killed by immersion in a water bath at 80° C for 75 min.

Alternatively, heat killed Listeria monocytogenes was obtained commercially

(Invitrogen) and was used according to the manufacturer‟s instructions.

Western blotting, isotopic labeling, and immunoprecipitation. For Western

blotting, 6 well dishes of cells were treated and infected as described in figure

legends. At times indicated, cells were washed with PBS and lysed in 1% SDS.

Protein concentration was determined by BCA assay (Pierce Chemicals) and

equivalent amounts of protein were analyzed by Western blotting with rabbit

antiserum against the SV5 NP, P or M proteins (28) or IRAK-M (Abcam). As a

loading control, blots were probed with an antibody against actin (citation

needed). Blots were visualized with horseradish peroxidase-conjugated

antibodies and enhanced chemiluminescence (Pierce Chemicals).

To examine protein synthesis, cells were mock infected or infected at a

high moi with virus as indicated in figure legends. At the time pi indicated in

figure legends cells were starved for 25 min with DMEM lacking cystein and

27

methionine and then radiolabeled for 2 hrs with 100 μCi/ml Tran[35S]-label.

Following radiolabel, cells were washed with PBS and lysed in 1% SDS. Equal

amounts of protein were immunoprecipitated using rabbit polyclonal antiserum to

PIV5 P proteins as described previously (28). Immunoprecipitated protein was

resolved on SDS-PAGE, which was dried and exposed to film.

Macrophage migration assays. Chemotaxis was evaluated using 24-well

Boyden chambers (BD Biosciences) with 3-μm pore polycarbonate filters as

previously described (33). Human recombinant M-CSF, RANTES/CCL5, VEGF,

or MCP-1/CCL2 (Invitrogen) were used at the final concentration of 100 ng/ml in

migration media (RPMI 1640 supplemented with 1% FBS). Macrophages were

resuspended in migration media and seeded at 2 x105 cells/well. Following

migration, filters were fixed and stained using Diff QUICK Stain kit (IMEB Inc.)

For each well, the number of migrated cells was calculated as the number of

cells counted in ten consecutive high power fields. All stimuli were assayed in

triplicates, and results were expressed as mean ± SD. To determine the effect of

TNF-α on migration, a neutralizing antibody against TNF-α (Biosource Inc.) or

IFN-β (PBL International) was added to cells immediately following infection at a

concentration of 10ug/mL. Exogenous TNF-α (Promega) was added to naïve

macrophages at a concentration of 100pg/mL.

Enzyme-linked immunosorbent assay, cell viability assays and IFN

signaling assays. Immunoreactive IL-6 or TNF-α (BD Opt EIA; BD

28

Biosciences), or IFN-β (PBL Biomedical Laboratories) or MIF (Ray Biotech) in

infected extracellular media was quantified by a dual antibody sandwich ELISA

according to manufacturer‟s protocols. To allow comparison between

experiments, cytokine levels were determined for the number of cells at the time

of infection and normalized to 106 cells. Cell viability was measured at the

indicated times pi by using a CellTiter 96 AQueaous One Solution cell

proliferation assay (Promega) according to the manufacturer‟s instructions. Data

are expressed as fold increase in cell viability relative to mock infected cells.

For IFN signaling assays, macrophages were treated with 103 IU of

exogenous type I IFN for twelve hours prior to infection with Vesicular Stomatitis

Virus (the kind gift of Doug Lyles) at moi 3. 24 h pi cell viability was measured as

described above.

Cell surface labeling, FACS, immunofluorescence and microscopy. For

detection of surface markers, cells were stained with the following conjugated

monoclonal antibodies according to manufacturer‟s recommendations; CD11c,

CD14, CD 64, CD 80, and Annexin V, all from BD Pharmigen. Samples were

analyzed on a FACScan Caliber instrument using Cell Quest software (Becton-

Dickinson).

Analysis of Mumps Virus NP protein was carried out by

immunofluorescence. Briefly, at 24 h pi cells were fixed in 100% ice-cold

methanol at 4°C, washed and incubated with a primary antibody against the MuV

NP protein (the kind gift of Tony Schmidt), and a secondary antibody conjugated

29

to a 488 fluorophore (Alexafluor). Samples were mounted using ProLong Gold

Antifade Reagent which includes DAPI (Invitrogen).

Phase and fluorescent microscopy were carried out as described

previously using a Nikon Eclipse microscope and a 20x lens (102). Images were

captured under phase contrast or visualized for GFP or DAPI expression using

Qimaging digital camera and processed using Q-capture software. Exposure

times were manually set to be constant between samples.

30

CHAPTER III

Mumps Virus Infection Inhibits Macrophage Chemotaxis in a TNF-α

Dependent Manner

(Sections of this chapter are being prepared for publication)

Human macrophages are restricted for migration following PIV5

infection. Extensive data in our laboratory and the laboratories of others has

characterized infection of epithelial cells with PIV5. However, less was known

about how WT PIV5 interacted with cells of the innate immune system. Thus, we

sought to characterize WT PIV5 infection in primary human macrophages. To

generate macrophages, CD14+ monocytes were isolated from human PBMC and

cultured for six days in the presence of MCSF. At that time, >90% were found to

be CD14+, CD11b+, and CD64+ by flow cytometry, a characteristic marker profile

of monocyte-derived macrophages (MDM, 14). Primary human macrophages

from a healthy donor were infected with WT PIV5 at a moi of 10. At 24, 72, and

120 h pi, cells were examined by microscopy for evidence of infection by GFP.

In Fig 5A, we observed that primary human macrophages were found to be GFP

positive at all time points tested, with no overwhelming evidence of CPE present

in the infected cells versus mock infected (data not shown). We also examined

the amount of the pro-inflammatory cytokine IL-6 produced by these cells during

WT PIV5 infection. Primary human macrophages were infected with WT PIV5 at

a moi of 10, and at 24, 72, 120, or 168 h pi supernatant was taken and used in

31

an ELISA specific for IL-6. While the cells were found to be productively infected

with WT PIV5, infected macrophages produced low levels of pro-inflammatory

cytokines such as IL-6 shown in Fig 5B, which is consistent with previous data in

our laboratory both with macrophages and epithelial cell lines (14, 102,104).

To confirm that infection of these cells with WT PIV5 was not inducing

high levels of CPE, primary human macrophages from three donors were

infected with WT PIV5 at a moi of 10. At 24 h pi, an MTT assay was done to

evaluate cell viability. As shown in Fig 5C, infected macrophages from all three

donors did not display significant decreases in cell viability at 24 h pi when

compared to mock infected cells.

While infection of macrophages with WT PIV5 did not appear to have

negative effects upon the cells, we hypothesized that viral infection may have

effects on macrophage functions such as migration. To test this hypothesis,

primary human macrophages were infected with WT PIV5 at a moi of 10.

Twenty-four h pi, cells were taken, counted, placed in a transwell and were

allowed to undergo chemotaxis towards PBS as a negative control, or towards

the chemokines MCP-1 or M-CSF for 4 hours. At the end of 4 hours, the

transwell filters were fixed and stained, and then the average number of cells per

10 fields visualized at 20x magnification was calculated. As shown in Fig 5D,

both mock and PIV5 infected cells had approximately equal low levels of

spontaneous migration towards PBS. However, significantly fewer PIV5 infected

cells migrated towards both MCP-1

32

Figure 5. PIV5 productively infects macrophages and inhibits their ability

to migrate.

Figure 5. A-B) Macrophages were mock infected or infected with WT PIV5 moi 10. At indicated times post infection viral replication was assayed by microscopy (panel A). Cell media was used in an ELISA for IL-6 production at times indicated. Amount of IL-6 was normalized to 106 cells, and is done in triplicate for one representative donor with standard error bars (panel B). C) Macrophages were mock infected or infected with WT PIV5 moi 10. 24 h pi an MTT assay was done to assay cell viability. D) Macrophages were mock infected or infected with WT PIV5 moi 10. 24 h pi cells were used in a migration assay towards indicated chemokine as described in Materials and Methods. Results are in triplicate with standard error bars for one representative donor. ** p≤0.005

0

20

40

60

80

100

120

Donor 1 Donor 2 Donor 3

% o

f M

ock

Infe

cte

d V

iabili

ty

A. B.

C.

PIV5-GFP

0

200

400

600

800

1000

1200

1400

Mock WT PIV5 P/V Mutant LPS

IL-6

pg/m

l per

10

6cells

D.

0

20

40

60

80

100

120

140

160

****

Control MCP-1 MCSF

Mock

Phase

GFP

Mean #

of cells

per

10 H

PF

Mock Infected PIV5 Infected

Day 1

Day 3

Day 5

33

and MCSF compared to mock infected cells. Taken together, these results

indicate that while WT PIV5 infection of macrophages appears to have little effect

on cell viability or cytokine production, it does impact the ability of the cells to

migrate towards chemoattractants.

Primary human macrophages are susceptible to infection with MuV.

We hypothesized that MuV would have similar effects on primary human

macrophages as seen with PIV5. To determine if macrophages were susceptible

to MuV infection, primary human macrophages were mock infected or infected

with PIV5 as a positive control or MuV at a moi of 10. 24 h pi, cells were

radiolabeled with 35S, and the P protein was immunopreciptated and visualized

on SDS-PAGE. As shown in Fig 6A, macrophages do appear to be susceptible

to infection with MuV, as evidenced by the band indicating the presence of the P

protein. To confirm that infection at a high moi resulted in the majority of the cells

being infected, we conducted immunofluorescence on infected cells. Primary

human macrophages were infected with MuV at a moi of 10. 24 h pi the cells

were fixed permeabilized and stained with an antibody against the NP protein

and visualized by microscopy. As shown in Fig 6B, the majority of the cells

present in the dish have detectable levels of the NP protein, indicating that the

majority of the cells are productively infected by MuV.

To determine the effects of MuV infection on macrophage viability, cells

were mock infected or infected with MuV at a moi of 10, and then used in an MTT

assay (Fig 6C) or cells were stained for Annexin V

34

Figure 6. MuV productively infects macrophages with little effect on

macrophage viability.

Figure 6. A) Macrophages were mock infected or infected with WT PIV5 or MuV moi 10. 24 h pi cells were radiolabeled and lysed, followed by immunoprecipitation for the viral P protein. B) Macrophages were mock infected or infected with MuV moi 10. 24 h pi cells were permeabilized and immunofluorescence was used to visualize the viral NP protein. C) Macrophages were mock infected or infected with MuV moi 10. At 24 h pi, an MTT assay was done for cell viability. Samples were run in triplicate with standard error bars and are shown for one representative donor. D) Macrophages were mock infected or infected with MuV moi 10. 24 h pi cells were stained for Annexin V and percentage of positive cells was analyzed by FACS. One representative donor is shown.

APIV5 MuV

P protein

MockB

10

20

30

40

50

60

70

80

90

100

Mock MuV

% A

nn

exin

V P

osit

ive

D

0

20

40

60

80

100

120

% m

oc

k v

iab

ilit

y

Mock

MuV

Mock

MuV

DAPI α-NP

C

35

and quantified by FACS (Fig 6D). As shown in Fig 6C, there was no significant

difference in cell viability at 24 h pi in MuV infected macrophages versus mock

infected macrophages. MuV-infected macrophage viability at 24 h pi was also

found to be similar to that of PIV5 infected macrophage viability at the same time

point (Fig 5C). MuV infected macrophages were also found to low levels of

annexin V positive cells, similar to what was seen in mock infected macrophages.

Taken together this data suggests that primary human macrophages are

susceptible to infection with MuV, and that this infection does not have a

significant impact on cell viability out to 48 h pi.

MuV infection inhibits macrophage migration to a variety of

chemoattractants in an MOI dependent manner. To determine if MuV

infection had an impact on the ability of macrophages to migrate towards

chemoattractants, primary human macrophages were infected with MuV at a moi

of 10. Twenty-four h pi the macrophages were counted and placed in transwells,

then allowed migrating towards M-CSF for 4 hours. At the end of 4 hours, the

transwell membranes were fixed and stained, and the average number of cells in

10 fields seen at 20x magnification was counted. As shown in Fig 7A, both mock

and MuV infected macrophages displayed low levels of spontaneous migration

towards PBS as a negative control. However, macrophages infected with MuV

were less able to migrate towards M-CSF compared to mock infected

macrophages. This is the same phenotype observed with WT PIV5 infected

macrophages shown above. To determine if migration was reduced only towards

36

M-CSF or if other known chemokines were also implicated primary human

macrophages were mock infected or infected with MuV at a moi of 10. 24 h pi

the cells were used in a migration assay as described above. The macrophages

were allowed to migrate towards PBS as a negative control, toward M-CSF as a

positive control, or towards several different chemokines which utilize receptors

different from M-CSF. Interestingly as shown in Fig. 7B, migration of MuV

infected macrophages was reduced towards all of the chemokines tested,

regardless of which receptor was targeted.

Once we had concluded that MuV infected macrophage migration was

reduced towards a variety of chemoattractants, we wanted to determine if the

inhibition of migration was dependent upon viral replication. To test this, primary

human macrophages were infected with MuV at increasing mois. Twenty-four h

pi the cells were used in migration assays as described previously, using M-CSF

as a chemoattractant. We observed in Fig 8A that the number of cells able to

migrate towards M-CSF decreased in a moi-dependent manner, with

macrophages infected at a low MOI more readily able to migrate compared to

cells infected at a moi of 25. After observing that the greatest difference

occurred using a moi of 25, we infected macrophages at that moi for the

remainder of our experiments.

We then hypothesized that viral replication would be required in

macrophages to inhibit migration towards chemokines. To test this, primary

human macrophages were infected with UV-inactivated MuV at a

37

Figure 7. MuV infection inhibits macrophage migration towards various

chemoattractants.

Figure 7. A-B) Macrophages were mock infected or infected with MuV moi 10. 24 h pi cells were used in a migration assay towards the indicated chemoattractant as described in Materials and Methods. Assay was done in triplicate with standard error bars. Results are shown for one representative donor. P values were calculated using Student‟s T test. ^ p≤0.05, *p≤0.001

A B

0

100

200

300

400

500

600

Control MCSF RANTES VEGF0

5

10

15

20

25

30

35

40

45

Me

an #

of ce

lls p

er

10 H

PF

Control MCSF

^

Mock

MuV

Mock

MuV

*

* *

Me

an #

of ce

lls p

er

10 H

PF

38

Figure 8. Inhibition of macrophage migration is exacerbated by viral load

and dependent on viral replication.

Figure 8. A) Macrophages were mock infected or infected with MuV at moi indicated. 24 h pi cells were used in a migration assay towards M-CSF as described in Materials and Methods. Results are in triplicate with standard error bars for one representative donor. P value was calculated using Student‟s T test. *** p≤0.001. B) Macrophages were mock infected or infected with UV-inactivated MuV at moi 10. 24 h pi cells were used in a migration assay towards M-CSF as described in Materials and Methods. Results are in triplicate with standard error bars for one representative donor.

A

0

5

10

15

20

25

30

35

40

45

50

# c

ells

/10

HP

F

Mock

MuV UV

None MCSF

B

0

50

100

150

200

250

300

350

# c

ells

/10

HP

F

Mock

MOI 1

MOI 5

MOI 25

MCSFNone

***

*** p ≤ 0.001

39

moi of 25. 24 h pi the macrophages were used in a migration assay as described

previously, using M-CSF as the chemoattractant. As shown in Fig. 8B, it appears

that while macrophages infected with the UV treated virus display some reduced

level of migration towards M-CSF, a greater number of virally treated

macrophages are undergoing chemotaxis, compared to macrophages infected

with MuV.

Taken together, these results indicate that MuV virus infection is sufficient

to reduce macrophage migration towards chemoattractants. This reduced

migration appears to occur regardless of which receptor the chemokine targets,

and occurs in a moi-dependent manner. Importantly, viral replication is required

for drastic inhibition of migration to occur.

Inhibition of infected macrophage migration occurs in a TNF-α

dependent manner. We hypothesized that a soluble factor may be responsible

for inhibiting macrophage migration. To test this, primary human macrophages

were infected with MuV at a moi of 25. Twenty-four h pi, media from these cells

was removed and UV treated to inactivate any live virions. This UV-inactivated

media was then added to fresh, uninfected macrophages overnight. Following

treatment, the macrophages were then used in a migration assay towards M-

CSF as described above. When the number of macrophages able to migrate

towards the M-CSF was analyzed, we observed in Fig 9A that both mock treated

and MuV media treated macrophages had similar levels of spontaneous

migration towards M-CSF. Remarkably, we found that macrophages treated with

40

media from MuV infected macrophages were deficient in their ability to migrate

towards M-CSF compared to untreated macrophages.

Several factors have been implicated in inhibiting macrophage migration.

One well known factor is macrophage inhibitory factor (MIF). MIF production is

thought to occur during infection to retain macrophages at the site of infection to

aid in clearance (17). To determine if MIF was being produced during MuV

infection of macrophages, primary human macrophages were mock infected, or

infected with MuV. 24 h pi media from infected cells was taken and used in an

ELISA specific for MIF. As shown in Fig 9B, mock infected macrophages

displayed low levels of MIF, which is consistent with reports that low levels of MIF

are constitutively produced by some cells such as macrophages (17). A549 cells

were infected with P/V-CPI-, a potent inducer of pro-inflammatory cytokines, as a

positive control. Interestingly, macrophages infected with MuV did not produce

levels of MIF over mock infected macrophages.

Soluble factors other than MIF are able to inhibit macrophage chemotaxis.

One cytokine that has been shown to inhibit migration in a MIF-independent

manner is TNF-α (37, 42). To determine if TNF-α was being produced by

macrophages during MuV infection, primary human macrophages were mock

infected or infected with MuV at a high moi. 24 h pi media from infected cells

was used in an ELISA specific for TNF-α. As shown in Fig 9C, mock infected

macrophages displayed low levels of TNF-α, although it should be noted that

donor to donor variability in levels of TNF-α secretion was observed.

41

Figure 9. Inhibition of migration during MuV infection is caused by a

soluble factor.

Figure 9. A) Macrophages were mock infected or infected with MuV moi 10. 24 h pi media from mock and infected cells was removed and UV-inactivated. Treated media was placed on uninfected cells overnight. Treated macrophages were then used in a migration assay towards M-CSF as described in Materials and Methods. Results are in triplicate with standard error bars for one representative donor. P value was calculated using Student‟s T test. *p≤0.001 B-C) Macrophages were mock infected or infected with MuV moi 10. 24 h pi media from infected cells was used in an ELISA specific for MIF (panel B) or TNF-α (panel C). Results are shown in triplicate with standard error bars for one representative donor, and were normalized for 106 cells.

0

500

1000

1500

2000

2500

MIF

(pg/m

l) p

er

10

6cells

Mock MuV P/V Mutant

Macrophage Inhibitory Factor

0

10

20

30

40

50

60

70

Mock MuV P/V Mutant

TN

F-

(pg/m

l) p

er

10

6cells TNF-

B C

A

0

20

40

60

80

100

120

140

160

180

*

Control MCSF

Mock Media

MuV Media

Mean #

of cells

per

10 H

PF

42

Interestingly, MuV infected macrophages had elevated levels of TNF-α

compared to mock infected macrophages or the positive control of A549 cells

infected with P/V-CPI-.

The production of TNF-α by macrophages has been shown to reduce or

inhibit migration (37). To determine if the production of TNF-α was responsible

for inhibiting chemotaxis in MuV infected macrophages, primary human

macrophages were mock infected or infected with MuV at a high moi. In the

replacement media following infection, an excess of a neutralizing antibody

against TNF-α or IFN-β as a control was added. 24 h pi the cells were used in a

migration assay as described previously. As shown in Fig 10A, MuV infected

macrophages had significantly reduced migration towards M-CSF, consistent

with previous data. Interestingly, macrophages treated with a neutralizing

antibody for TNF-α migrated towards M-CSF at levels similar to mock infected

cells. We also noted that mock infected macrophages treated with a neutralizing

antibody against TNF-α displayed an increased level of migration compared to

mock treated, mock infected macrophages. This effect was not seen in

macrophages which were treated with a neutralizing antibody against IFN-β, as

shown in Fig 10B. MuV infected macrophages treated the neutralizing antibody

against IFN-β displayed a reduced level of migration, similar to that seen in non-

treated, infected macrophages.

43

Figure 10. Neutralization of TNF-α is necessary to restore migration in MuV

infected macrophages.

Figure 10. A-B) Macrophages were mock infected or infected with MuV moi 10. In replacement media following infection cells were treated with PBS or a neutralizing antibody against TNF-α (panel A) or IFN-β (panel B). 24 h pi cells were used in a migration assay towards M-CSF as described in Materials and Methods. Results are shown in triplicate with standard error bars for one representative donor. P value was calculated using Student‟s T Test. # p≤0.002, *p≤0.01

0

100

200

300

400

500

600

700

Mean #

of cells

per

10 H

PF

Mock MuV

Anti-TNF-α

#

0

50

100

150

200

250

300

Mock MuV

Untreated

MCSF

PBS

Mock MuV MuV +

Anti-IFN

MCSF

PBS

Mean #

of cells

per

10 H

PF

A. B.

* *

44

To determine if exposure to TNF-α alone was sufficient to inhibit

macrophage chemotaxis, primary human macrophages were treated overnight

with exogenous TNF-α in the absence of viral infection, at levels similar to the

amount of TNF-α produced during MuV infection seen in Fig 9C. After treatment

with TNF-α, the macrophages were used in a migration assay as described

above. As shown in Fig 11, the addition of exogenous TNF-α was sufficient to

inhibit the migration of mock infected macrophages.

Taken together, these results indicate that the production of TNF-α by

macrophages during MuV infection is sufficient to inhibit macrophage migration,

and that neutralizing TNF-α restores the ability of infected macrophages to

migrate. This effect appears to be specific to TNF-α, as treatment with a

neutralizing antibody specific to IFN-β is not sufficient to restore the ability of

macrophages to migrate. The addition of exogenous TNF-α to un-infected

macrophages is also sufficient to inhibit macrophage migration, further

supporting these results. This supports a model that viral of induction of TNF-α

alone is sufficient to inhibit macrophage migration, and that viral infection is not

required to inhibit migration.

45

Figure 11. TNF-α is sufficient to inhibit chemotaxis in uninfected

macrophages.

Figure 11. Macrophages were treated with PBS or exogenous TNF-α as described in Materials and Methods. After overnight exposure, macrophages were used in a migration assay towards M-CSF as described in Materials and Methods. Results are shown in triplicate with standard error bars for one representative donor. P value was calculated using Student‟s T test. ***p≤0.001

0

10

20

30

40

50

60

70

80

90

100

PBS Treated TNF- Treated

***

MCSF

PBS

Mean #

of cells

per

10 H

PF

46

CHAPTER IV

Activation of Macrophages by Bacterial Components Relieves the

Restriction on Replication of an Interferon-Inducing Parainfluenza Virus 5

(PIV5) P/V Mutant

Sections of this chapter have been previously published

(Briggs et. al., 2011, Microbes and Infect.)

The P/V-CPI- mutant is restricted for growth in primary human

macrophages due to the presence of IFN-beta. Replication of WT PIV5 and

the P/V-CPI- mutant was analyzed in primary human macrophages. Cells were

infected at moi of 10 with WT PIV5 and or the P/V-CPI- mutant, both of which

express green fluorescence protein (GFP) as an additional gene between HN

and L (102). At 18 hours post infection (h pi), infection was assayed by

microscopy and western blotting with antibodies to PIV5 NP and P. As shown in

Fig. 12A, primary human MDM were susceptible to infection with WT PIV5,

evidenced by the large number of GFP-positive cells. By contrast, cells infected

with P/V-CPI- displayed far fewer GFP-positive cells. The low level of infection by

the P/V mutant was supported by results from western blotting for viral proteins.

As shown in Fig 12B, MDM infected with WT PIV5 displayed much higher levels

of viral protein compared to cells infected with the P/V mutant. Infection of

47

macrophages was not able to be driven by infecting the cells with P/V-CPI- at a

higher MOI, as shown in Figure 12C.

In epithelial and fibroblast cell lines, we have shown that the P/V-CPI-

virus is a potent inducer of pro-inflammatory cytokines and type I IFN (34, 102)

and that growth of the P/V mutant is partially sensitive to IFN (105). As shown in

Fig. 13A, MDM infected with the P/V mutant secreted higher levels of IFN-beta

than cells infected with WT PIV5. To determine the role of secreted IFN-beta on

susceptibility of MDM to the P/V mutant, primary human MDMs were infected

with P/V-CPI- at moi 10, and media were supplemented with PBS as a control, or

with a neutralizing antibody against IFN-beta or TNF-alpha as a control. At 18 h

pi, cells were visualized by microscopy to detect GFP expression. As shown in

Fig. 13B, P/V-CPI- infected cells treated with a neutralizing antibody against IFN-

beta display increased numbers of GFP-positive cells compared to mock or

control antibody treated cells.

Taken together, these results indicate that P/V-CPI- is restricted for

infection of primary human MDM, this restriction cannot be overcome by

increasing moi, and that IFN-beta secreted in response to P/V-CPI- infection is at

least partially responsible for restricted replication.

Stimulation of macrophages with heat killed bacteria enhances the

susceptibility of human MDM to P/V-CPI- infection. MDMs generated by

culturing with MCSF are considered to be in a naïve state (39). Macrophages can

48

Figure 12. The P/V Mutant is restricted for replication in primary human

macrophages.

Figure 12. A-B) MDM were mock infected or infected at an moi of 10 with WT PIV5 or P/V-CPI- and cells were assayed at 24 h pi by microscopy for GFP expression (panel A), or by western blotting with antibodies specific for the indicated proteins (panel B). C) MDM were infected with P/V-CPI- at increasing mois as indicated. 24 h pi cells were assayed by microscopy for GFP expression.

Mock WT PIV5 P/V-CPI-

Phase

GFP

A. Mock WT P/V-CPI-

Actin

P

NP

MOI 20

MOI 40

MOI 200

C

B.

49

Figure 13. IFN-β production by MDM contributes to the restriction of the P/V Mutant. Figure 13. A) MDM were mock infected or infected with WT PIV5 or the P/V mutant at moi 10. 24 h pi media were assayed by ELISA for levels of IFN-beta. Cytokine production was normalized to 106 MDM. Results are the mean values (with standard error bars) from three samples from a representative donor. B) MDM were infected at a moi of 25 with P/V-CPI- and then incubated in media supplemented with PBS as a control, or with antibody specific for TNF-alpha or IFN-beta. Cells were examined by microscopy at 18 h pi for GFP expression.

0

40

80

120

160

200

240

280

Mock WT PIV5 P/V-CPI-

PBS Anti-IFN- ab

IFN

-beta

(pg/m

L p

er

10

6ce

lls)

Phase

GFP

A

BAnti-TNF- ab

50

be activated upon encountering pathogens, which results in increased levels of

cell surface markers such as CD80, and the secretion of pro-inflammatory

cytokines such as IL-6 (65). Given that the phenotype of activated macrophages

differs significantly from naïve cells, we tested the hypothesis that macrophages

activated by exposure to stimuli such as bacteria would display enhanced

susceptibility to infection with the P/V mutant. Primary human MDM were

stimulated overnight with a panel of heat killed Gram positive bacteria, including

Listeria monocytogenes (LM), Streptococcus pyogenes (GAS), and Bacillus

anthracis (BA) and levels of CD80 at 18 h post exposure were determined by

flow cytometry. As shown in Fig. 14A for MDM from two individual donors,

exposure of MDM to heat killed LM (HKLM) or GAS (HKGAS) resulted in more

cells expressing high levels of CD80. Similarly, MDM exposed to heat killed LM,

GAS, or BA secreted very high levels of IL-6 at 24 h post exposure (Fig 14B).

Importantly, MDM exposed to these heat killed bacteria did not secrete IFN-beta

(Fig 14C), consistent with previous data (90). These results indicate that MDM

exposed to heat killed bacteria are converted from a naïve to an activate state.

To determine if the P/V-CPI- mutant was restricted for replication in

activated cells, MDM were treated with PBS or exposed to heat killed LM, GAS,

or BA for 12 hours. Bacteria were removed, and cells were washed before

infection at a moi of 25 with P/V-CPI-. At 18 h pi, cells were examined by

microscopy for levels of GFP-positive cells. As shown in Fig. 15A, the number of

GFP-positive MDM in cultures stimulated with heat killed LM, GAS, or BA prior to

infection was dramatically higher compared to PBS-treated cells. Fig 15B shows

51

Figure 14. Activation of primary human macrophages following treatment

with heat killed bacteria.

Figure 14. Human MDM were treated overnight with PBS or with 108 cfu of heat killed Listeria monocytogenes (LM), Streptococcus pyogenes (GAS), or Bacillus anthracis (BA). At 12 h p exposure, cells were analyzed by flow cytometry for levels of CD80 (panel A). Media were assayed by ELISA for levels of IL-6 (panel B) and IFN-beta (panel C). Cytokine production was normalized to 106 MDM. Panels A and B show data from two donors; panel C is a typical result from a representative donor.

0

1

2

3

4

5

6

7

8

Mock LM GAS BA

IL-6

(ng/m

L p

er

10

6cells

)

Donor 1

0

2

4

6

8

10

12

IL-6

(ng/m

L p

er

10

6cells

)

Donor 2

Mock LM GAS BA0

50

100

150

200

250

IFN

-beta

(pg/m

L p

er

10

6cells

)

Mock LM GAS BA P/V

A.

B. C.

5

10

15

20

25

30

35

% C

ells

CD

80

HKLM

Treated

HKGAS

Treated

Mock

Donor 1

Mock HKLM

Treated

% C

ells

CD

80

10

20

30

40

50

60

70

80

90

HKGAS

Treated

Donor 2

52

flow cytometric quantification of the percent of GFP-positive cells and mean

fluorescence intensity (MFI) for infected cells from a typical donor after treatment

with heat killed Gram positive bacteria. Enhanced viral gene expression in

activated MDM was not limited to GFP. This is evident in Fig. 15C, where

western blotting of cell extracts from MDM exposed to HKLM prior to infection

had increased levels viral NP, P and M compared to PBS-treated infected

macrophages. GFP expression was consistently found to be the highest for cells

that were treated with heat killed GAS, but was observed with all Gram positive

bacteria tested so far. Enhanced GFP expression was seen following treatment

of cells for as little as one hr before P/V mutant infection. Enhanced virus gene

expression was not seen when MDM were treated with live Gram positive

bacteria, or when MDM were treated with heat killed Gram negative bacteria (e.g.

Haemophilus influenzae and Bordetella pertussis) or cytokines (e.g. IL-1beta,

TNF-alpha ;) (Figure 16). To determine if monocytes or MDM early in the

differentiation process were also susceptible to enhanced P/V mutant infection

following activation, CD14+ monocytes were treated with PBS or HKLM as

described previously. Monocytes or day 1 (D1) MDM were then infected with the

P/V mutant at a high MOI, and the number of GFP positive cells was quantified

24 h pi. As shown in Fig. 17, naïve monocytes or D1 MDM treated with PBS

were resistant to infection with the P/V mutant. However, following stimulation

with HK LM, monocytes and D1 MDM showed enhanced susceptibility to P/V

mutant infection. Taken together, these data indicate that activated macrophages

are more susceptible to infection with P/V-CPI- compared to naïve macrophages,

53

and that this activation is specific to stimulation with heat killed Gram positive

bacteria prior to viral infection.

Bacteria-induced enhancement of P/V mutant infection is transient.

The differences in susceptibility of naïve and activated macrophages to P/V

infection raised the question of whether this was a permanent or transient

property of cells exposed to heat killed bacteria. To test this, MDM were treated

with heat killed LM for 3 hrs, bacteria were removed and the cells were washed

twice with PBS. MDM were then infected with the P/V mutant at an moi of 25 at

0, 4, 12, 24, and 30 h post removal of the HKLM. At 24 h pi of each culture, cells

were examined for GFP expression by microscopy and FACS. As shown in Fig.

18A for cells from a representative donor, MDM were highly susceptible to

infection with the P/V mutant at 0 and 4 h post removal of HKLM. However, by 12

h post removal of HKLM, the number of cells susceptible to infection with the P/V

mutant had decreased to levels seen with MDM treated with PBS alone. When

quantified by FACS, ~40% of cells were GFP-positive at 0 and 4 h post removal

of HKLM, with an MFI of ~25 and 15, respectively. However, by 12 h post

removal of HKLM, less that 10% of cells were found to be GFP-positive, and the

MFI of these cells was similar to that of PBS-treated control cells. These results

indicate that the susceptibility of MDM to P/V infection that is induced by HKLM

treatment is transient in nature, lasting up to 12 hrs post removal of HKLM.

54

Figure 15. The P/V-CPI- mutant is not restricted for replication in primary

MDM that have been activated by exposure to Gram positive bacteria.

Figure 15. A) GFP expression. Primary human MDM were treated with PBS or 108 cfu of heat killed LM, GAS or BA. MDM were washed and infected with P/V-CPI- at an moi of 25. Cells were examined by microscopy at 18 h pi for GFP expression. B) Quantification of GFP-expressing cells. Macrophages treated as described for panel A were analyzed by FACS to determine the number and mean fluorescent intensity (MFI) of GFP positive cells. Results are representative of 3 independent experiments with three different donors. C) Western Blotting. Primary MDM were treated with PBS or 108 cfu/mL of heat killed LM for 12 h prior to infection with P/V-CPI- at an moi of 25. Cell lysates were harvested at 18 h pi and analyzed by western blotting with antibodies specific for PIV5 NP, P, and M proteins, and cellular actin.

NP

Actin

P

Actin Actin

M

PBS HK LM HK GAS HK BA

0

20

40

60

80

PBS LM GAS BA

% G

FP

positiv

e c

ells

0

2

4

6

8

10

12

MF

I F

old

over

Mock

PBS LM GAS BA

A. B.

C. PBS HKLM PBS HKLM PBS HKLM

Phase

GFP

Phase

GFP

Donor

#1

Donor

#2

55

Figure 16. Alternately activated MDMs are not susceptible to infection with

the P/V Mutant

Figure 16. A) MDMs were stimulated with PBS, HK LM as a positive control, or live S. pyogenes (GAS), L. monocytogenes (LM), B. pertussis (BP), or H. influenzae (HI) before being infected with the P/V mutant moi 10. 24 h pi cells were assayed for GFP production by microscopy. B) MDMs were stimulated with heat killed Gram negative bacteria before being infected with the P/V mutant moi 10. 24 h pi cells were assayed for GFP production by microscopy. C) MDM were stimulated with exogenous cytokines before being infected with the P/V mutant moi 10. 24 h pi cells were assayed for GFP production by microscopy.

PBS HK LM GAS LM BP HI

Phase

GFP

A

B

PBS IFN-γ IL-1β TNFα

Phase

GFP

HK BP HK LM

GFP

HK HI

Phase

PBS

C

56

Figure 17. Monocytes display enhanced susceptibility to P/V Mutant

infection following HKBac stimulation.

Figure 17. A-B) CD14+ monocytes or D1 MDM were treated with PBS or

HKLM as described previously, before infection with the P/V mutant moi 25. 24 h pi % of GFP positive cells (panel A) or MFI (panel B) was quantified using FACS. Results are for one representative donor.

0

10

20

30

40

50

60

70

80

90

100

% G

FP

Po

sit

ive

Mock

PV+PBS

PV+HKLM

Naïve Monocytes D1 Macrophages

0

2

4

6

8

10

12

14

16

18

Fo

ld o

ver

Mo

ck M

FI

PV+PBS

PV+HKLM

Naïve monocyte D1 Macrophage

A

B

57

MDM that are activated with heat killed Gram positive bacteria

maintain IFN signaling, but produce lower levels of IFN-beta in response to

P/V-CPI- infection. The above finding that the P/V mutant is restricted for

replication in MDM by IFN-beta raised the hypotheses that treatment of MDM

with heat killed bacteria altered either the production of IFN or IFN signaling. To

determine if the IFN signaling pathway was altered by exposure to heat killed

bacteria, we tested whether activated MDM were protected by exogenously

added IFN from killing by VSV challenge. MDM were treated with either PBS or

HKLM for 3 h as described previously, washed to remove bacteria and then

treated with exogenous IFN-beta overnight to induce an anti-viral state. These

cells were infected with VSV at an moi of 3, and viability was determined at 24 h

pi. As shown in Fig. 19A for two donors, cells that did not received exogenous

IFN had viability that was reduced following VSV infection (black bars). VSV

reduced viability to only ~30-40% of control cells, consistent with the previous

finding that macrophages generated with MCSF are partially resistant to killing by

VSV (30). Reduced viability was also seen in cells treated with HKLM, consistent

with a lack of IFN induction by HKLM treatment. Most importantly, IFN treatment

protected cells from VSV infection (hatched bars, Fig. 19A), with increased

viability seen in the case of PBS as well as HKLM-treated MDM.

To determine levels of IFN-beta produced by infected MDM that were

activated with heat killed Gram positive bacteria, MDM were treated with PBS as

a control, or with heat killed LM, GAS or BA and then washed and infected with

P/V-CPI- at an moi of 25. At 24 h pi, levels of secreted IFN-beta were assayed by

58

Figure 18. The increased susceptibility of activated MDM to P/V-CPI-

infection is transient.

Figure 18. A) P/V-CPI- replication levels after removal of heat killed LM. Primary human MDM were treated with PBS or 108 cfu of heat killed LM for 12 hours, after which the bacteria was removed, cells were washed and replaced in fresh media. At the indicated times post removal of bacteria, MDM were infected with P/V-CPI- at an moi of 25. At 18 h pi with the P/V mutant, cells were examined by microscopy for number of GFP-positive cells. Panels B and C show FACS analysis to quantify the percentage of GFP-positive cells (panel B) and the MFI of the GFP-positive cells (panel C). Results are representative of two independent experiments with two donors.

Mock

Infected

P/V-CPI-

Infected

Time of P/V-CPI- Infection Post Removal of HKLM

0 4 12 24 30

0

10

20

30

40

50

P/V-CPI-

HKLM-

Treated

% G

FP

Positiv

e

0 hr

4 hr

12 hr

24 hr

30 hr

P/V-CPI-

PBS-

Treated

Mock

Infected

A.

B.

0

10

20

30

40

50

60

0 hr

4 hr

12 hr

24 hr

% G

FP

Positiv

e

P/V-CPI-

HKLM-

Treated

P/V-CPI-

PBS-

Treated

Mock

Infected

Donor

#2Donor

#1

59

an ELISA. As shown in Fig. 19B for cells from three individual donors, infection of

PBS-treated MDM with P/V-CPI- resulted in increased levels of IFN-beta,

consistent with results in Fig. 12D above. By contrast, MDM exposed to heat

killed bacteria prior to infection with P/V-CPI- produced background levels of IFN-

beta similar to that seen in the absence of virus infection. For one sample (donor

1), IFN-beta was induced by infected MDM that had been treated with GAS, but

this was not consistently seen for cells from a number of other donors. Taken

together, these data are consistent with the proposal that the increased

susceptibility of activated MDM to the P/V mutant is due to decreased production

of IFN following virus infection and not due to alterations in IFN signaling.

Elevated levels of IRAK-M in MDM that are activated with heat killed

Gram positive bacteria. In some cases, macrophages activated by bacterial

components have been shown to subsequently become refractory to further

stimulation, and this is thought to be at least in part from the upregulation of

inhibitory molecules that dampen potential hyperactive responses (1, 59). We

have tested the hypothesis that P/V-CPI- infection of MDM that have been

treated with HKLM results in elevated expression of negative regulators of anti-

microbial responses. MDM were treated with PBS as a control or with HKLM and

then infected at an moi of 25 with P/V-CPI-. Cell extracts were analyzed by

western blotting for levels of IRAK-M, an inhibitor of TLR signaling and IFN

induction (53). As shown in Fig 20 for two separate donors, IRAK-M levels were

elevated in the infected MDM that had been activated by prior exposure to HKLM

60

compared to the PBS-treated control. Given that IRAK-M targets IRAK-1 for

inhibition and IRAK-1 can be involved in the induction of IFN-beta (53, 83), our

data are consistent with a model whereby activation of MDM by bacterial

components leads to elevated IRAK-M and a reduction in the capacity of these

activated MDM to respond to P/V-CPI- infection.

61

Figure 19. MDM that are activated with heat killed Gram positive

bacteria maintain IFN signaling, but produce lower levels of IFN-beta in

response to P/V-CPI- infection.

Figure 19. A) IFN-beta signaling in HKLM-treated MDM. Primary human MDM were treated PBS, HKLM or HKGAS. Cells were then washed and treated with PBS or 100 IU of IFN-beta for 12 hr. Cells were then infected with VSV at an moi of 3 and cell viability was determined 24 h pi as described in Materials and Methods. Results are the mean values (with standard error bars) from three samples from a representative donor. # p≤0.006 and * p≤0.002. B) IFN-beta secretion from activated MDM. Primary human MDM were treated with PBS or the indicated heat killed bacteria as previously described, and then infected with P/V-CPI- at an moi of 25. Media were analyzed at 24 h pi by ELISA for levels of IFN-beta. Results from four individual donors are shown. *p≤0.006, **p≤0.002.

100

150

200

250

300

350

450

IFN

-beta

(pg/m

L p

er

10

6ce

lls)

PBS HK LM PBS HK LM HK GAS HK BA

Mock Infected P/V-CPI- Infected

* ** 350

50

0

Donor 1

Donor 2

Donor 3 *

Treatment

Before Infection

B.

0

20

40

60

80

100

120

140

PBS HKLMPBS

+IFN-β

HKLM

+IFN-β

% v

iab

le c

ells

#

A. 200

150

100

50

0PBS HKGASPBS

+IFN-β

HKGAS

+IFN-β

% v

iab

le c

ells

Donor 4

*

62

Figure 20. IRAK-M is induced to higher levels in P/V-CPI- infected MDM that

have been activated by exposure to bacterial components.

Figure 20. MDM from two donors were treated overnight with PBS or HKLM prior to infection with P/V-CPI- at an moi of 25. At 18 h pi, cell lysates were analyzed by western blotting for levels of IRAK-M or actin as a load control.

Actin

IRAK-M

PBS HKLM PBS HKLM

Donor #1 Donor #2

P/V Mutant Infected

Actin

IRAK-M

Donor #1

Mock Infected

PBS HKLM

63

CHAPTER V

DISCUSSION

Host responses to microbial invasion are thought to primarily be of benefit

to the host. The ability to control and eliminate viral and bacterial infection is

crucial for the survival of the host. However, recent published data and work in

this thesis indicate that these host defense mechanisms may inadvertently be

beneficial to the pathogens, instead of detrimental. In this thesis, I have

demonstrated two mechanisms by which Rubulaviruses are able to exploit host

cell responses to increase their own survival and replication.

One mechanism depicts how MuV infection of primary human

macrophages inhibits the ability of these cells to undergo chemotaxis. During

viral infection of macrophages and of other cells, such as epithelial cells, the host

cell produces cytokines to combat the infection and reduce viral load. In the case

of MuV infection in macrophages I have shown that robust amounts of TNF-α are

produced. TNF-α is known to be an important regulator for immune cells. It has

the ability to induce apoptotic cell death, induce inflammation, and inhibit viral

replication (32). During MuV infection of macrophages, the production of TNF-α

results in the paralysis of these cells as evidenced by an inability of these cells to

migrate towards a gradient of chemokine. This inability to migrate may lead to

the reduction of other macrophage functions such as phagocytosis, may lead to a

delayed adaptive immune response, and may increase the inflammatory

64

environment surrounding the infections, all of which are beneficial for MuV

replication and proliferation.

In a second mechanism, macrophages are rendered more susceptible to

infection with P/V-CPI- following exposure to bacterial components. Detection of

pathogens such as bacteria by macrophages leads to activation of these cells.

These activated macrophages are more cytotoxic, and have increased

production of pro-inflammatory cytokines, all of which should be beneficial to the

host and prevent spread of disease (65). In this thesis, I have shown that

activation of macrophages by bacterial components actually relieves the

restricted replication of P/V-CPI- in those cells. While macrophages exposed to

bacterial components become activated and develop enhanced microbial

responses, these responses must be tightly regulated to avoid host damage.

When the host upregulates factors to control innate immune responses, it begins

to reduce those inflammatory responses. We propose that this negative

regulation provides a “window of opportunity” in which the host is actually more

susceptible to infection with viruses such as P/V-CPI-. This window provides an

opportunity for the virus to productively infect the host, and the host is unable to

defend itself against secondary infections.

Susceptibility of macrophages to infection with WT PIV5 and MuV. I have

demonstrated that macrophages are permissive to WT PIV5 and MuV infection.

Infection of macrophages by PIV5 and MuV does not appear to have significant

impact on cell viability or cytokine production during infection, at least during the

65

first 24-48 h pi. However, the ability of infected macrophages to migrate towards

chemokines is impaired. This impairment occurs with both PIV5 and MuV

infected macrophages, although the effect is more pronounced in MuV infected

cells. This inhibition of migration is independent of chemoattractant, and requires

a productive viral infection. No detectable level of MIF is present during MuV

infection, which suggests that this inhibition is not occurring by MIF-induced

paralysis. However, MuV infected macrophages do display increased levels of

TNF-α during viral infection. Importantly, neutralization of the TNF-α produced

during MuV infection is sufficient to relive the restriction on migration in

macrophages.

Our results here with WT PIV5 and MuV infected macrophages

demonstrate that the virus in these cells behaves similarly to viral infection of

epithelial cells. While much is known about infection of Rubulavirus in epithelial

cell lines, less is known about viral infections in innate immune cells such as

macrophages. WT PIV5 has long been shown to be able to establish productive,

long term infections in human epithelial cell lines with minimal cell death, and

poor induction of pro-inflammatory cytokines and type I IFN (22, 102). PIV5

infected macrophages displayed robust viral protein production, low levels of

CPE, and poor induction of pro-inflammatory cytokines such as IL-6 and IFN-β

(14). By contrast, primary human monocyte-derived dendritic cells, which are

derived from the same progenitor cell as macrophages displayed increased

levels of cell death while still being a poor inducer of pro-inflammatory cytokines

and type I IFN when infected with WT PIV5 (6). Human primary pDCs, which are

66

also important for innate immune responses, were also found to be susceptible

to infection with WT PIV5 following high moi infection, however viral infection of

these cells lead to increased levels of type I IFN, specifically IFN-α (63). Based

on the results in this thesis, as well as published data, it appears that primary

human macrophages are more phenotypically similar to human epithelial cells

during Rubulavirus infection.

Effects of WT PIV5 and MuV infection on macrophage function. As

discussed earlier in this thesis, one of the most important functions of a

macrophage is its ability to migrate. Macrophages use chemotaxis for the

induction of innate and adaptive immune responses. Macrophages are recruited

to the site of infection by chemokines. Once they have reached the site of

infection, macrophages become activated and begin to phagocytose pathogens,

and release cytokines to combat infection and recruit additional cells.

Macrophages at the site of infection are also able to uptake antigen and migrate

to lymph nodes to present this antigen to T cells to stimulate an adaptive immune

response. Deficiencies in macrophage chemotaxis can lead to increased

inflammation, and a delayed adaptive immune response (65).

We hypothesized that this apparently benign infection of WT PIV5 and

MuV in macrophages may have effects upon macrophage function; specifically

the ability of the cells to migrate towards chemoattractants. As shown in Fig 5D

and Fig. 7A, macrophages infected with WT PIV5 and MuV at a high moi were

resistant to migration towards M-CSF. Inhibition of migration was found to be

67

greater in MuV infected macrophages than during PIV5 infection. The receptor

which detects M-CSF and MCP-1 is a homodimeric type III receptor tyrosine

kinase (39). The receptor for VEGF is also a tyrosine kinase receptor (43).

However, other chemoattractants such as RANTES are detected by G-protein

coupled receptors (GPCRs) (68) and migration of infected macrophages towards

RANTES is also impaired (Fig. 7B).

Our data could be explained by the downregulation of chemokine

receptors following MuV infection. However, analysis of the GPCRs CCR1 and

CCR5 on the surface of mock or MuV infected macrophages indicates that

infected cells actually have higher levels of these receptors, which are used to

detect RANTES, compared to mock infected cells (data not shown). This

indicates that downregulation of chemokine receptors is most likely not the cause

for decreased migration in infected macrophages. It is also possible that

dysfunctional rearrangement of the actin cytoskeleton of infected macrophages is

responsible for decreased chemotaxis during infection. However, analysis of the

actin cytoskeleton using phalloidin displayed no obvious differences in

morphology of mock infected versus MuV infected macrophages (data not

shown). This indicates to us that the inhibition in the migration pathway occurs

after receptor-ligand binding, but before changes in polarity would be noted.

The role of MIF in inhibiting macrophage chemotaxis. Our data indicate that

a soluble factor produced during MuV infection is responsible for inhibiting the

migration of macrophages. This is evident in Fig. 9 where UV-treated

68

supernatant from infected macrophages is sufficient to inhibit migration in

uninfected macrophages. Previous results have indicated that MuV infection of

cells leads to the secretion of factors that inhibit macrophage migration. Fluid

taken from the parotid glands of chimpanzees showing clinical symptoms of MuV

infection was treated to neutralize any infectious virions, before being added to

naïve macrophages. Following treatment, these macrophages were found to be

deficient in their ability to migrate towards chemoattractants. Due to the recent

discovery of the chemokine MIF, this behavior was characterized as “MIF-like”,

although the identity of the secreted factor was never determined (31, 111).

From this, we can hypothesize that in these previous studies TNF-α produced

during viral infection may be contributing towards the loss of macrophage

migration.

Our results indicate that the secreted factor by MuV in primary human

macrophages is not MIF, as shown in Fig 9B, where MuV infection of

macrophages did not elicit levels of MIF above those produced by mock infected

cells. This indicates to us that MIF is not responsible for the inhibition of

macrophage migration during MuV infection. This result differs from those

obtained using human cytomegalovirus (HCMV) (33). In that study, elevated

levels of MIF produced during viral infection were shown to be responsible for the

loss of chemotaxis. However, these results were dependent upon the production

of virally induced MIF. The addition of exogenous MIF did result in inhibition of

migration through the downregulation of receptors, which did not occur during

HCMV infection (33).

69

Why is HCMV capable of inducing MIF and MuV appears to be deficient in

MIF induction? HCMV is a double stranded DNA virus, and as such is most

likely detected by different host cell receptors compared with MuV, which is a

negative strand RNA virus. While MIF was one of the first cytokines identified,

detailed understanding of regulatory and induction pathways is not well

understood (8). While cells such as macrophages have been shown to express

MIF constitutively, increased production of MIF has been noted in cells following

TLR stimulation or exposure of cells to LPS (16, 17). One hypothesis for the

induction of MIF by HCMV and not by MuV could be explained by the ability of

HCMV to signal through TLR2 and CD14 (23). CD14 has been shown to be a

receptor for LPS binding and signaling (98). It stands to reason that detection of

HCMV by TLR2 and CD14, both of which are expressed on the surface of

macrophages, would thereby lead to an increased production of MIF during viral

infection. However, MuV is most likely detected by different cellular receptors.

We know that Rubulaviruses such as PIV5 are detected by cytoplasmic RIG-I,

and not MDA-5 or TLR3 in epithelial cells (64). While we do not know the viral

detector for Rubulaviruses in macrophages, macrophages do express

cytoplasmic sensors such as RIG-I (data not shown) and I hypothesize that a

similar mechanism is used to detect Rubulaviruses in macrophages. These RNA

sensors are not associated with increased MIF secretion, thus explaining the

differences in cytokine upregulation between these two viruses.

70

Upregulation of TNF-α during MuV infection. One of the most interesting

results in examining the inhibition of macrophage migration following MuV

infection was the increase in TNF-α production during viral infection, and its role

in inhibiting chemotaxis. As shown in Fig. 9C, macrophages infected with MuV

displayed increased levels of TNF-α compared to mock infected macrophages.

Interestingly, our data indicates that the production of TFN-α during MuV

infection is necessary to inhibit macrophage migration. As shown in Fig. 10A,

primary human macrophages treated with a neutralizing antibody against TNF-α

immediately following MuV infection regained the ability to migrate towards M-

CSF. This was not seen in macrophages treated with a neutralizing antibody

against IFN-β (Fig. 10B). Additionally, treatment of uninfected macrophages with

exogenous TNF-α for 12 hours was sufficient to inhibit the ability of those cells to

migrate towards M-CSF (Fig. 11).

Some labs have claimed that Rubulaviruses inhibit the induction of TNF-α

through the viral SH protein (61, 109). The SH protein is a 44 amino acid

membrane associated protein expressed on the surface infected cells and virions

(41). In these studies the SH protein of WT PIV5 was shown to be responsible

for resisting TNF-α induced apoptosis, by preventing the induction of TNF-α

during viral infection (61). Indeed, PIV5 is a poor inducer of all pro-inflammatory

cytokines including TNF-α in murine and bovine epithelial cell lines. MuV is

closely related to PIV5, and is hypothesized to function similarly to PIV5 in

primary human epithelial cell lines. The MuV used in our studies is the Enders

strain, which is an attenuated vaccine strain of MuV frequently used in laboratory

71

studies since its isolation (29). Data obtained by others demonstrated that the

SH protein of MuV Enders strain was sufficient to block TNF-α production when

inserted into the backbone of PIV5 and independently transfected into cells

(109). However, our data indicates that TNF-α production is strongly upregulated

during MuV infection, and upregulated compared to mock during PIV5 viral

infection (Fig 9B, data not shown). As both of these viruses have intact SH

proteins, how is TNF-α induction occurring?

One explanation is the difference in signaling pathways between human

epithelial cell lines and primary human macrophages. Human epithelial cell lines,

specifically those which are tumor-derived may lose or downregulate their

responses to pathogens, leading to decreased production of pro-inflammatory

cytokines such as TNF-α. Primary cells derived from a human donor may not

have lost these signaling pathways, and as such can respond to pathogens in a

way that more accurately mimics that of the host. Additionally, it could be the

difference between viral infections of epithelial cells versus macrophages.

Macrophages, being cells involved in innate and adaptive immune responses,

may be more sensitive to pathogen infection, or may have different mechanisms

of detecting viral infection compared to epithelial cells, which leads to increased

production of cytokines such as TNF-α.

What is the mechanism by which TNF-α is able to inhibit macrophage

migration? Figure 21 depicts a working model of our hypothesis. Several studies

have demonstrated that the treatment of macrophages with TNF-α is sufficient to

inhibit macrophage migration (37, 42.). While the signaling pathway which leads

72

to macrophage migration is not well understood, a potential model can be

deduced from what is known about the signaling pathway of other migratory

cells. The migration of smooth muscles, eosinophils, and endothelial cells was

found to require signaling through the mitogen-activated protein kinase (MAPK)

pathways. These kinases are threonine/serine specific protein kinases which act

in a cascade manner through phosphorylation (77). Increased levels of MAPK

phosphorylation is seen in macrophages exposed to the chemoattractant MCP-1

(37). However, macrophages exposed to TNF-α displayed low to no detectable

levels of MAPK phosphorylation as soon as 30 minutes after treatment. Instead,

TNF-α induced increased levels of MAPK phosphatase 1 (MKP-1) (37). MKP-1

has been shown to be a negative regulator of MAPK signaling, and function by

de-phosphorylating MAPK (21, 86). We hypothesize that secretion of TNF-α by

macrophages during MuV infection is then able to signal on cells in an autocrine

or paracrine fashion, and upregulates the production of MKP-1. When infected

macrophages are subjected to stimuli by chemoattractants, the elevated levels of

MKP-1 prevent MAPK phosphorylation, preventing downstream NFκB activation

and subsequently, macrophage chemotaxis. Work to determine levels of MKP-1

in MuV infected macrophages is ongoing.

73

Figure 21. A model for inhibition of migration by TNF-α.

Figure 21. A) When chemoattractants bind their receptors, downstream signaling occurs, resulting in MAPK phosphorylation, and the activation and nuclear translocation of NFxB. This nuclear translocation results in macrophage migration. B) When TNF-α is produced during MuV infection in macrophages, it is able to signal via its receptor on the cell surface. TNF-α signaling results in the upregulation of the phosphatase MPK-1, which acts by dephosphorylation MAPK. This prevents the activation and nuclear translocation of NFxB upon chemokine binding, and inhibits macrophage migration.

Physiological consequences of inhibited macrophage migration. Tissue

macrophages are recruited to sites of infection, where their migration is then

halted and they become activated through pro-inflammatory cytokine production

by other cells, and through encountering pathogen. Their function then shifts

from migration to uptake of pathogens, antigen processing, and cytokine

Figure 21. A) When chemoattractants bind their receptors, downstream signaling occurs, resulting in MAPK phosphorylation, and the activation and nuclear translocation of NFxB. This nuclear translocation results in macrophage migration. B) When TNF-α is produced during MuV infection in macrophages, it is able to signal via its receptor on the cell surface. TNF-α signaling results in the upregulation of the phosphatase MPK-1, which acts by dephosphorylation MAPK. This prevents the activation and nuclear translocation of NFxB upon chemokine binding, and inhibits macrophage migration.

Cell membrane

cytoplasm

nucleus

MAP3K

MAP2K

MAPK

NF κ B

NF κ B MIGRATION

A B

MAP3K

MAP2K

MAPK

NF κ B

NF κ B

X

TNF - a

MPK - 1

MPK - 1

X

chemoattractants chemoattractants

74

Physiological consequences of inhibited macrophage migration. Tissue

macrophages are recruited to sites of infection, where their migration is then

halted and they become activated through pro-inflammatory cytokine production

by other cells, and through encountering pathogen. Their function then shifts

from migration to uptake of pathogens, antigen processing, and cytokine

production. However, like all immune functions of the host, these functions need

to be tightly regulated to prevent host damage. When macrophage migration is

inhibited, macrophages are unable to egress from sites of infection, which leads

to increased cytokine production, increased recruitment of additional cells, and

increased inflammation at sites of infection. We propose that this work may have

important clinical implications for understanding the pathology of disease

progression in viruses which cause chronic inflammation, such as MuV. Active

MuV infection is characterized by increased swelling and inflammation of the

parotid glands, testes in males, and CNS where the virus is able to localize and

productively infect (49). These increased inflammatory conditions may serve to

enhance viral persistence.

Susceptibility of macrophages to infection with P/V-CPI-. My work has also

demonstrated that activated macrophages are more susceptible to infection with

P/V-CPI- compared to naïve macrophages. Given the dramatic differences in

replication of the parainfluenza virus mutant P/V-CPI- in human epithelial cells

(102) versus immature DC (6), it was important to determine the susceptibility of

primary human macrophages to P/V-CPI-. Our results indicate that the P/V-CPI-

75

mutant is restricted for replication in naïve MDMs due to the induction of IFN-

beta. Our most striking finding came from our work addressing the question of

whether P/V-CPI- was also restricted in activated macrophages. Our work

demonstrates that the restriction on P/V-CPI- replication can be transiently

alleviated by prior activation of MDMs with heat killed Gram positive bacteria.

Increased susceptibility of the MDM that are exposed to Gram positive bacteria is

linked to their limited ability to produce IFN-beta in response to infection with P/V-

CPI-, while the ability of these activated MDM to respond to exogenous IFN-beta

remains intact. As detailed below, these data suggest a model whereby exposure

to Gram positive bacteria transiently suppresses the ability of MDM to mount an

IFN response that normally restricts P/V-CPI- replication.

Effect of bacterial components on viral replication. Our most striking finding

is that P/V-CPI- replication is no longer restricted in MDM that have been

activated by exposure to a range of heat killed Gram positive bacteria such as

Listeria monocytogenes (LM), Streptococcus pyogenes (GAS), and Bacillus

anthracis (BA). The ability of cells to change susceptibility to virus infection after

exposure to bacterial components has been reported previously for HIV infection

of macrophages (4, 96, 113). Similarly, recent work from Nguyen et al. (69) has

shown that RSV replication is enhanced when immature DC or primary epithelial

cells are treated with the TLR2 agonist Pam3CSK. Our results with the

parainfluenza virus PIV5 differ substantially from these other reports in several

regards. First, MDM infection by P/V-CPI- is not enhanced by pretreatment with

76

individual TLR 2 agonists such as Pam3CSK4 as shown for HIV infections (data

not shown) (4, 47) Additionally, enhancement of P/V replication is seen following

HKLM treatment of immature DC (not shown) and naïve MDM, but not in

epithelial cell lines such as A549 cells. Thus, the mechanism behind enhanced

P/V-CPI- replication appears to have different characteristics compared to other

systems. However, enhancement of P/V-CPI- replication was found to occur

following prior treatment of MDM with heat killed bacteria, and during

simultaneous addition of HKBac, which corresponds with enhanced RSV

replication following simultaneous addition of virus and stimulant (69).

What is the bacterial component that serves as an activator of MDM to

increase susceptibility to P/V-CPI- infection? MDM susceptibility to P/V-CPI- is

not enhanced by exposure to Gram negative bacteria such as Haemophilus

influenzae and Bordetella pertussis (Fig. 16). This is most likely due to LPS from

Gram negative bacteria stimulating type I IFN production through activation of

TLR 4 (1, 10) and the subsequent induction of an antiviral state in the MDM.

While the addition of LPS has been shown to suppress the production of IFN by

influenza virus, viral growth was not inhibited because the virus was able to block

IFN signaling and continue replication (62), the P/V mutant lacks the ability to

degrade STAT1 and inhibit IFN signaling (102). This inability to block IFN

signaling likely contributes to the restricted viral growth in macrophages treated

with heat killed Gram negative bacteria. By contrast, bacterial lipoprotein

signaling through TLR2 does not induce IFN-beta. Bacterial lipoproteins have

been identified in multiple species of Gram positive bacteria (112), are released

77

from bacteria when they are damaged or lysed, and have been shown to be

present in abundance in preparations of heat killed bacteria. Bacterial

lipoproteins have been shown to be capable of activating macrophages and

repressing macrophage responses to infection (112).

The role of IFN-β in limiting viral infection. Our results here with restricted

replication of P/V-CPI- in MDM contrast sharply with previous results in epithelial

cells. While replication of P/V-CPI- in epithelial cells is sensitive to added IFN, the

main effect of IFN is a slight delay and lower overall titers (103). However, under

high moi conditions P/V-CPI- replicates in epithelial cells faster and to higher

levels than WT PIV5 (102). By contrast, P/V-CPI- replication in primary human

macrophages is strongly restricted such that very few GFP-positive cells are

seen during high moi infection. Thus, the strength of the antiviral response or the

landscape of IFN-inducible genes may differ considerably between epithelial cells

which are largely permissive to P/V-CPI- and MDM which strongly restrict

replication. Consistent with this proposal, Newcastle Disease Virus (NDV) has

been shown to be restricted for growth in human macrophages and this

correlated with high levels of antiviral gene products such as RIG-I, IRF-3 and

IRF-7 (108). It is very likely that the ability of IFN to block detectable GFP

expression in infected MDM reflects a restriction at the level of secondary viral

transcription, since this is the step in the virus lifecycle that gives rise to abundant

and detectable viral gene expression (55). Previous work has shown that addition

of IFN to Vero cells infected with a related P/V mutant (CPI-) substantially

78

changed the steepness of the gradient of viral RNA transcription, with a high “fall

off” of transcription at the P-M junction and longer poly A tails on M mRNA (19).

This raises the hypothesis that the rapid IFN response induced by P/V-CPI-

restricts replication in MDMs at least in part by altering the processivity of the viral

polymerase and the steepness of the 3‟-5‟ gradient of transcription. Consistent

with this proposal, our western blotting (Fig. 15C) shows a greater effect of MDM

activation on enhancing the expression of 3‟ distal genes (e.g. M protein) versus

3‟ proximal genes (e.g. NP). The ability of type I IFN to limit early steps of the

viral life cycle in macrophages has been characterized for other viruses. Human

Immunodeficiency Virus-1 (HIV-1) has also been shown to be restricted for

growth in human macrophages due to the induction of type I IFN during early

stages of the viral infection (25).

Mechanism for the inhibition of IFN-β induction. What is the mechanism by

which MDMs that are activated by bacterial components are more susceptible to

P/V-CPI- infection? Several results are not consistent with alterations at an early

step in the virus lifecycle such as attachment or fusion at the plasma membrane.

First, increasing the moi to 200 did not increase susceptibility. Secondly,

neutralization of extracellular IFN increased susceptibility to infection. Since IFN

induction by PIV5 is dependent on virus replication (102, 103), this supports a

mechanism that is post entry. Finally, MDM that are activated by heat killed

bacteria secrete less IFN in response to P/V-CPI- infection. Thus, our data

support a model whereby activation of MDM by bacterial components renders

them less able to mount an IFN response to P/V-CPI- infection.

79

Figure 22 depicts a model for how the block in IFN induction is occurring.

Our data could be explained by the principle that pathogen-induced signaling is

tightly regulated by feedback mechanisms in order to avoid damage to the host

through uncontrolled pro-inflammatory cytokine secretion (47). One method of

downregulating antimicrobial cascades involves the production of cellular factors

such as IRAK-M which function to negatively regulate signaling (47, 101). Unlike

other members of the IRAK family, IRAK-M is expressed exclusively in

macrophages, lacks kinase activity and negatively regulates signaling following

TLR2, TLR4 and Nod2 stimulation by preventing the phosphorylation of IRAK-1

and its subsequent detachment from MyD88 (53, 40, 67, 106). Published data

indicate that repeated and prolonged stimulation of Nod2 with MDP, a

component of peptidoglycan which is found in Gram positive bacteria, results in

upregulation of IRAK-M in primary human macrophages (40). Additionally,

ssRNA generated during paramyxovirus infection has been shown to induce IFN-

beta in a Nod2-dependent fashion (83). Thus, we hypothesize that P/V-CPI- may

be detected by Nod2 in primary macrophages, resulting in IFN-beta induction.

However, in macrophages that have been activated by heat killed bacteria,

subsequent P/V-CPI- infection provides additional Nod2 stimulation that results in

upregulation of IRAK-M, and this feeds back to inhibit further Nod-2 mediated

IFN-beta production. In epithelial cell lines, we have previously shown that P/V-

CPI- induces pro-inflammatory cytokines and IFN-beta through RIG-I and the

production of viral dsRNA (64). There are examples of a link between RIG-I and

IRAK-1 in VSV infections of macrophages (46). Thus, while it is unclear whether

80

detection of P/V-CPI- in primary macrophages is through Nod2 or RIG-I, a role

for IRAK-1 in either of these sensing pathways would be consistent with our

findings of upregulated IRAK-M, reduced IFN-beta secretion and enhanced P/V-

CPI- replication.

81

Figure 22. A Model for HKBac Enhanced Viral Infection in Macrophages

Figure 22. P/V mutant infection is normally detected by a cytoplasmic sensor. In epithelial cells this sensor is RIG-I; in macrophages the sensor is still unknown although both RIG-I and NOD2 are candidates. Detection of the virus leads to nuclear translocation of NFκB, AP-1 and IRF downstream of IRAK-1 signaling. This nuclear translocation upregulates IFN-β. However, in macrophages activated by HKBac prior to infection, an upregulation of IRAK-M occurs, which then acts upon IRAK-1, and prevents the induction of IFN-β upon subsequent viral infection.

82

Physiological implications of bacterially-enhanced viral infections. Our

results have implications for the design of attenuated IFN-sensitive RNA viruses

as therapeutic vectors or vaccine candidates (100). In order to improve safety

and reduce virulence, many of RNA virus vectors are being engineered to be

sensitive to IFN through alterations to viral antagonists of host cell responses.

Our P/V-CPI- mutant which both induces IFN and fails to block IFN signaling is

an example of this strategy for attenuation and is being developed as a vaccine

vector (6, 18) and for oncolytic therapy (34). Our results with the PIV5 P/V mutant

raise the possibility that under some in vivo conditions, viruses that are

engineered to be attenuated to improve safety may have enhanced replication

and virulence in cells exposed to bacterial components that suppress production

of IFN. Clinically, co-infections of the respiratory tract involving Gram positive

bacteria and negative strand RNA viruses such as the parainfluenza viruses are

well documented (e.g. 56), and our work highlights the need to understand the

extent to which one organism attenuates or accentuates replication of the other

organism.

Future Directions. The work described in this thesis identifies two unique

mechanisms by which Rubulaviruses take advantage of normal host cell

response to enhance their own ability to replicate and spread. We have

identified these two mechanisms as the induction of TNF-α by MuV to suppress

macrophage chemotaxis and inhibition of IFN-β induction during P/V mutant

infection in macrophages following stimulation of the cells with bacterial

83

components. However, there is still additional work to be undertaken to help

understand both of these mechanisms.

MuV inhibition of chemotaxis. While I have shown that TNF-α is both

necessary and sufficient to inhibit migration in MuV infected macrophages, many

questions still remain. I have shown that TNF-α is produced by primary human

macrophages during infection. This production of TNF-α is most likely due to

detection of the virus by the host cell. How is this virus detected? Currently, the

sensor of MuV infection in primary human macrophages is unknown.

Presumably MuV is detected by a cytoplasmic host cell sensor. This is the most

likely hypothesis because the addition of UV inactivated virus does not lead to an

inhibition of macrophage chemotaxis. Due to the similarity between WT PIV5

and MuV, I would hypothesize that MDA-5 is not responsible for the detection of

MuV, because of the functional V protein that MuV encodes. It would be useful

to knock down cellular receptors such as MDA-5, RIG-I, and Nod2 in

macrophages using siRNA to determine what is responsible for detecting MuV

upon entry into the host cell.

We have hypothesized that one model by which the inhibition of

chemotaxis is occurring is due to the presence of MKP-1 during MuV infection.

This model is depicted in Fig. 21. However, we do not know if MKP-1 is being

upregulated in macrophages infected with MuV. Through Western blotting, we

could determine if MKP-1 is upregulated during MuV infection, and when this

upregulation occurs. It would also be useful to know if MKP-1 is active, and what

84

it is specifically targeting. While we know that the MAPK are presumably

dephosphorylated by MKP-1, we do not know which MAPK in particular is

targeted.

I have also speculated in this document that inhibition of migration by MuV

would lead to a delayed adaptive immune response. However, we have not

assayed whether or not these MuV infected cells are deficient in their ability to

process and present antigen, or if they are less able to interact with T cells than

their uninfected counterparts. The ability to present antigen on their cell surface,

and the ability to interact and stimulate T cell proliferation could be assayed in

vitro. It may be that infected macrophages are just as proficient at antigen

processing and presentation as uninfected macrophages. This would indicate

that a lag in adaptive immune responses may be specifically due to the inability

of these cells to migrate to lymph nodes. Alternatively, the result may show that

macrophages infected with MuV are not only deficient in their ability to migrate,

but are also deficient in their ability to stimulate adaptive immune responses.

Either outcome would add to our knowledge of the effect of MuV infection on

innate immune responses.

Bacterial components enhance replication of the P/V Mutant. I have shown

in this thesis that macrophages stimulated with bacterial components are more

susceptible to infection with a P/V mutant, and that this enhanced viral replication

is caused by low induction of IFN-β. However, there are additional experiments

that could be carried out to increase our understanding of this mechanism.

85

Similar to the work of MuV infection in primary human macrophages that I

described above, we do not know the host cellular receptor for the P/V mutant in

primary human macrophages. In the model described in Fig. 22, I hypothesize

that either RIG-I or Nod2 is responsible for detecting the P/V mutant. Knock

down of both of these targets using siRNA followed by viral infection would help

us to determine which of these factors is responsible for detecting the P/V

mutant. We also do not know the receptor for the heat killed Gram positive

bacteria in macrophages. In this thesis, I have hypothesized that Nod2 may be

responsible for detecting heat killed bacteria in macrophages. Alternately, TLR2

may play a role in detecting the HKBac, or a combination of receptors may be

involved. Stimulation of the macrophages with a synthetic TLR2 ligand did not

enhance P/V mutant infection. Stimulation of macrophages with varying

combinations of TLR ligands also did not enhance P/V mutant infection. Thus, I

hypothesize that Nod2 may be playing a role.

It is also unclear what bacterial component is required for stimulation of

the macrophages and enhancement in P/V mutant infection. I have determined

that the factor responsible for enhanced P/V mutant infection is found in

association with the bacteria and is not a soluble factor. Stimulation of

macrophages with the supernatant from heat killed bacteria does not result in

enhanced P/V mutant infection. However, I have not determined if the

cytoplasm, periplasm, or cell wall is responsible for stimulation of the

macrophages. To test this hypothesis, Gram positive bacteria could be

fractionated, and the separate fractions could be added to the macrophages prior

86

to infection with the P/V mutant. Alternately, it could be that a specific

component of the bacterial cell wall such as lipotechoic acid or peptidoglycan is

required. Further work is still needed to elucidate this.

87

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CURRICULUM VITAE

CAITLIN MATTOS BRIGGS

ADDRESS: Department of Microbiology and Immunology Wake Forest University School of Medicine Medical Center Boulevard Winston-Salem, NC 27157 Telephone: (336) 716-9093 Email: cmattos@wfubmc.edu EDUCATION: 2011 Ph.D. (Microbiology and Immunology) Wake Forest University Winston-Salem, NC 2006 B.S. (Biology) East Carolina University Greenville, NC GRADUATE RESEARCH EXPERIENCE: 2006-2011 Graduate Student

“The Effects of Rubulavirus Infection on Macrophage Function.” Dr. Griffith D. Parks Wake Forest University School of Medicine

UNDERGRADUATE RESEARCH EXPERIENCE: 2004-2006 ECU Undergraduate Research Assistantship

“Quantifying and Qualifying the Bacterial and Fungal Loads on the termite Nasutitermes corniger.” Dr. John W. Stiller East Carolina University

2002-2004 ECU Undergraduate Research Assistantship “Genetic Mapping of the Red Alge Porphyra.” Dr. John W. Stiller East Carolina University

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TEACHING EXPERIENCE: Feb. 2011, Co-instructor, Advanced Topics in Microbiology Sept. 2010 Apr. 2010, Co-instructor, Fundamentals of Virology Nov. 2009 Apr. 2008 DNA Day Ambassador HONORS AND AWARDS: 2010 Thoyd Melton Award North Carolina Regional American Society for Microbiology 2010 Travel Award 29th American Society for Virology 2007-2008 Co-Chair

Wake Forest University Graduate Student Association

2006-2007 North Carolina Association of Rescue and

Emergency Medical Services Scholarship 2006 Detlev M. Bunger Scholarship 2005 Charles Bland Scholarship 2003-2006 East Carolina University Honors Program

Fellow 2002-2006 East Carolina University Honors Program

Research Assistantship PROFESSIONAL AFFILIATIONS:

2009-present American Society for Microbiology Student Member

2008-present American Society for Virology Student Member 2005-2006 Association of Southeastern Biologists Student

Member

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PUBLICATIONS:

Briggs CM, and Parks GD. 2011. Mumps Virus Infection Inhibits Macrophage Migration in a TNF-α Dependent Manner. In preparation. Briggs CM, Holder RC, Reid SD, Parks GD. 2011. Activation of Human Macrophages by Bacterial Components Relieves the Restriction on Replication of an Interferon-Inducing PIV5 P/V Mutant. Microbes Infect. 4:359-68. Manuse MJ, Briggs CM, Parks GD. 2010. Replication-Independent Activation of Human Plasmacytoid Dendritic Cells by the Paramyxovirus SV5 Requires TLR7 and Autophagy Pathways. Virology 405:383-89. Mattos C, Postava-Davignon M, Rosengaus R, Stiller JW. 2006. Estimation and Identification of Bacterial and Fungal Loads in the Termite Species Nasutitermes corniger. Explorations: The Journal of Undergraduate Research and Creative Activities for the State of North Carolina. 1:107-21.

FORMAL PRESENTATIONS:

Briggs CM and GD Parks. Stimulation of Macrophages with Gram Positive Bacteria Enhances Susceptibility to Infection with a PIV5 P/V Mutant. 2011. 10th St. Jude Children’s Hospital National Graduate Student Symposium. Briggs CM and GD Parks. Stimulation of Macrophages with Gram Positive Bacteria Enhances Susceptibility to Infection with a PIV5 P/V Mutant. 2010. North Carolina Regional American Society for Microbiology Meeting. Briggs CM and GD Parks. Stimulation of Macrophages with Gram Positive Bacteria Enhances Susceptibility to Infection with a PIV5 P/V Mutant. 2010. 29th American Society for Virology Annual Meeting, Montana State University Briggs CM and GD Parks. Stimulation of Macrophages with Gram Positive Bacteria Enhances Susceptibility to Infection with a PIV5 P/V Mutant. 2010. 11th Southeastern Regional Virology Conference.

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COPYRIGHT INFORMATION

Part of the work presented in Chapter IV of this thesis was reprinted from Microbes and Infection, 13, Briggs, C.M., Holder, R.C., Reid, S.D., and G.D. Parks. 2011. Activation of human macrophages by bacterial components relieves the restriction on replication of an Interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant, pp. 359-368. Copyright © 2011, permission from Elsevier.