The Effect of Androstenediol on the Influenza Virus APR8/34 in A549 Cells THESIS Presented in Partial Fulfillment of the Require

Home , Antigenic drift, Viral matrix protein

The Effect of Androstenediol on the Influenza Virus APR8/34 in A549 Cells

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Scott Franklin Wray, B.A.

Graduate Program in Pathology

The Ohio State University

2012

Master's Examination Committee:

W. James Waldman, Advisor

John F. Sheridan

Scott Franklin Wray

2012

Abstract

A major problem in fighting influenza virus infections with the current trivalent vaccines

and antivirals is the inherent genetic instability of the RNA-encoded proteins of the virus.

Current vaccines induce neutralizing antibodies specific for two major surface antigens

expressed on the virion. These two proteins are encoded by the hemagglutinin and

neuraminidase genes respectively, and in these genes, there is a high rate of point

mutation resulting in antigenic drift of the expressed proteins. A similar problem arises

with antivirals where resistant strains develop or are selected for when antivirals are used

in a widespread fashion. In other words, the virions that are sensitive to the antiviral are

killed, whereas those that are resistant to the antiviral are free to infect susceptible

individuals. Therefore, it would be beneficial to develop anti-influenza strategies that do not rely on treatments directed against the variable genes encoded by the virus. Based on previous work from our laboratory, we hypothesize that androstenediol inhibits influenza replication by modifying the host cell’s interaction with the virus. Our proposal is that treatment with androstenediol (AED), may lead to an approach that is not directed at the variable genes encoded by the viruses. Our results suggest that AED treatment may inhibit viral replication, as indicated by the influenza viral matrix protein, M1, gene expression. Additionally, our results exclude certain cellular mechanisms and suggest other possible mechanisms that will need to be examined in more detail.

Dedication

This document is dedicated to my family and friends.

iii

Acknowledgments

I would like to acknowledge all my advisors, fellow laboratory members, and other faculty members for their guidance, encouragement, and support during the course of my training.

Vita

May 1999 ...... Liberty Union-Thurston High

2003...... B.A. Biology, Capital University

2003 to 2004 ...... Graduate Research Associate, Department

of Integrated Biomedical Science, The Ohio

State University

2004 to 2009 ...... Graduate Research Fellow, Department of

Oral Biology, The Ohio State University

2009 to 2011 ...... Graduate Research Associate, Department

of Integrated Biomedical Science, The Ohio

State University

2011 to present ...... Master’s Student, Department of Pathology,

The Ohio State University

Publication(s)

1. Integrin engagement increases histone H3 acetylation and reduces histone H1 association with DNA in murine lung endothelial cells. Molecular Pharmacology. 68(2):

439-46, 2005.

Fields of Study

Major Field: Pathology

Table of Contents

Abstract ...... ii

Dedication ...... iii

Acknowledgments...... iv

Vita ...... v

List of Tables ...... iix

List of Figures ...... x

List of Abbreviations ...... xii

Chapter 1: Introduction ...... 1

Chapter 2: Androstenediol Suppresses Influenza Virus-Encoded M1 Gene Expression in

Human Respiratory Epithelial Cells ...... 19

Introduction ...... 19

Methods ...... 23

Results ...... 28

Discussion ...... 31

Chapter 3: Androstenediol’s Effect on Cellular Signaling and a Coalescing of Pattern

Recognition Receptors ...... 42

vii

Introduction ...... 42

Methods ...... 44

Results ...... 48

Discussion ...... 51

Chapter 4: Determining Whether Androstenediol Modifies Influenza Virus-Encoded M1

Gene Expression Through the Androgen Receptor ...... 57

Introduction ...... 57

Methods ...... 59

Results ...... 66

Discussion ...... 69

Chapter 5: General Discussion...... 80

References ...... 91

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List of Tables

Table 2.1. Primer and TaqMan Probe Sequences of cytokines M1 and 18S for Real Time

PCR ...... 35

Table 3.1. PCR array analysis of AED activated genes in A549 cell culture with exposure to influenza virus Primer and TaqMan Probe Sequences of cytokines M1 and

18S for Real Time PCR ...... 56

List of Figures

Figure 2.1 Dose Curve of AED and its Effect on Influenza Virus M1 Gene Expression in

A549 Lung Epithelial Cells ...... 36

Figure 2.2 Time Course of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells ...... 37

Figure 2.3 Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells

...... 38

Figure 2.4 Multiple Steroid Agonists and Antagonists Effect on Influenza Virus M1

Gene Expression in A549 Lung Epithelial Cells ...... 39

Figure 2.5 The above images were used to determine whether AED had an effect on influenza viral entry ...... 40

Figure 2.6 The above images were used to determine whether AED had an effect on influenza viral entry ...... 41

Figure 3.1 Data of Interferon Beta Gene Expression in A549 Lung Epithelial Cells ..... 53

Figure 3.2 Focused microarray analysis of AED activated genes in A549 cell culture without exposure to influenza virus ...... 54

Figure 3.3 Data of Interferon Beta Gene Expression in A549 Lung Epithelial Cells With

Exposure to Influenza Virus ...... 55

Figure 4.1 Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells

Treated With a Dose Curve of Methyltrienolone (R1881) ...... 74 x

Figure 4.2 Time Course of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells ...... 75

Figure 4.3 Data of Androgen Receptor Transient Transfection Assay With a Dose Curve

Comparing AED and DHT ...... 76

Figure 4.4 Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells

Treated With a Dose Curve of Cyproterone Acetate in an Inhibition Assay of Androgen

Receptor ...... 77

Figure 4.5 Both blots used varying concentrations of AR siRNA ...... 78

Fig 4.6 Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells

Treated With either control siRNA or androgen receptor ...... 79

List of Abbreviations

β-Me β-mercaptoethanol

ΔCT delta (change) cycle threshold

ΔΔCT delta delta cycle threshold

nM nanomolar

ng nanogram

µg microgram

µl microliter

18S 18S ribosomal RNA

A549 human lung adenocarcinoma epithelial cell line

AED 5-androstene-3ß-17ß-diol

AET 5-androstene-3ß-7ß-17ß-triol

AKT serine/threonine protein kinase

ANOVA One way or one factor analysis of variance

APR8 A/Puerto Rico/8

AR androgen receptor

ARE(s) androgen response element(s)

ATII(s) alveolar type II epithelial cell(s)

BC Before Christ

C Celsius

CA cyproterone acetate

xii

CARD caspase activation and recruitment domain

cDNA complimentary DNA

CO2 carbon dioxide

Cort corticosterone

COS7 African green monkey kidney fibroblast-like cell line

CT cycle threshold

DAPI 4',6-diamidino-2-phenylindole

DE dimethyl sulfoxide/ethanol

DEX dexamethasone dH2O distilled water

DHEA dehydroepiandrosterone

DHT dihydrotestosterone

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid dsRNA double-stranded RNA

Ebp1 ErbB3-binding protein eIF-2α eukaryotic initiation factor 2 alpha

ErbB3 epidermal receptor tyrosine kinase

ERK extracellular signal-related kinase

ETOH ethanol

FAM reporter dye 6-carboxyfluorescein

FBS fetal bovine serum

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Flut flutamide

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GR glucocorticoid receptor

HA hemagglutinin

HAU(s) hemagglutinin assay unit(s)

HEPES 4-(2-hydoxyethyl)-1-piperazineethanesulfuonic acid

HF hydroxyflutamide

HRP horseradish peroxidase

Hsp90 heat shock protein 90

IFN(s) interferon(s)

IFNAR interferon-α/β receptor

IgA immunoglobulin A

IgG immunoglobulin G

IL-1 interleukin-1

IL-2 interleukin-2

IPS-1 IFN-β promoter stimulator 1

IRAK interleukin receptor-associated kinase

IRF3 interferon regulatory factor 3

IRF7 interferon regulatory factor 7

Kb(s) kilobase(s)

LSD least significant difference

M molar (moles/liter)

xiv

M1 matrix protein

M2 ion channel; matrix protein 2

MDA5 melanoma differentiation-associated gene 5

ml milliliter

mRNA(s) messenger RNA(s)

MyD88 myeloid differentiation primary response gene (88)

NA neuraminidase

NF90 NFAT factor

NFκB nuclear factor κB

NFAT nuclear factor of activated T-cells

nm nanometer

NO nitric oxide

NOS2 nitric oxide synthase 2

NP nucleoprotein

NR(s) nuclear receptor(s)

NS1 nonstructural protein 1

NS2 nonstructural protein 2

OAS 2’-5’ oligoadenylate synthetase

PA polymerase acidic protein

PAMPs pathogen-associated molecular patterns

PB1 Polymerase basic protein 1

PB1-F2 polymerase base fragment 2

PB2 Polymerase basic protein 2

PBS Phosphate buffered saline

PCR polymerase chain reaction

p.i. post infection

PI3K phosphatidylinositol-3-kinase

PKR RNA-dependent protein kinase

poly I:C polyinosine-polycytidylic acid

PRRs pattern recognition receptors

pUC18 plasmid cloning vector

R1881 methyltrienolone

RAW 264.7 mouse macrophage cell line

RIG-I retinoic acid inducible gene-I

RLR(s) RIG-I-like receptor(s)

RNA ribonucleic acid

RNAase ribonuclease

RPMI 1640 Roswell Park Memorial Institute 1640 rRNA ribosomal RNA

TAM 6-carboxy-tetramethyl-rhodamine

TANK traf family member-associated NFκB activator

T cell thymus derived cell

TLR(s) toll-like receptor(s)

TLR3 toll-like receptor 3

xvi

TLR7 toll-like receptor 7

TLR8 toll-like receptor 8

TNFα tumor necrosis factor alpha

TRAF6 tumor necrosis factor receptor-associated factor 6

TRAIL TNF-related apoptosis inducing ligand

TRIF TIR-domain-containing adapter-inducing interferon-β

VIC 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein

xvii

Chapter 1: Introduction

1.1 Overview

Today’s influenza viruses are the evolutionary products resulting from the interactions between the host and this genetically unstable virus. In spite of the development of reasonably effective vaccines, influenza infection remains a major health concern globally. For example, in the United States, even during a thirty year period when a major pandemic did not occur, from 1976 to 2006 23,607 deaths occurred each year from influenza-associated deaths (CDC 2010). This should evoke the question as to how the influenza virus can be such a health threat in the era of effective vaccines. Two types of vaccines for influenza virus are currently used, a live, cold-adapted attenuated influenza virus vaccine and a trivalent inactivated vaccine. Both typically contain three influenza viral strains, A(H3N2), A(H1N1), and B, and both induce increased plasma cell production of neutralizing IgA and IgG antibodies specific for the hemagglutinin and neuraminidase of the viral strains used in the vaccines (Cox et al. 2004). However, because the segmented influenza virus RNA (ribonucleic acid) genome is highly amenable to genetic mutation, from antigenic shift and drift, antigenically distinct subtypes emerge each year, requiring adjustment of the vaccine. Even more alarming, antigenic shift can result in the emergence of new viral strains, such as the avian influenza virus and the swine influenza virus, for which humans lack adequate immunity.

The emergence of such strains has recently prompted fears of impending pandemics with

catastrophic implications.

In addition to antigenic drift and antigenic shift, which involve modifications of

the virus’ genes that result in protein modification, the virus can further evade innate

immune responses by sequestering and disguising internal structures such as viral dsRNA

(double-stranded RNA) (Short 2009). This, in effect, helps hide the virus from detection by host pattern recognition receptors. Furthermore, the influenza virus has also adapted itself to use the host advantageously to facilitate its own proliferation. For example, by exploiting a common cytokine secondary signaling messenger (phosphatidylinositol-3-

kinase [PI3K]) the virus can actually prevent apoptosis aimed at killing the virus before

its budding from a cell (Ehrhardt et al. 2007). Such evolutionary adaptations regularly

displayed by the influenza virus indicate a need for a treatment that does not focus on the

virus but on the host, making it much more difficult for the virus to simply evade by

mutation.

One treatment that has shown potential to protect animals with an influenza

infection is androstenediol, AED. In fact, studies have been performed in which mice

were infected with 24 HAUs (hemagglutinin assay units) of influenza virus APR8

(A/Puerto Rico/8) after treatment with AED or DMSO (dimethylsulfoxide)/ethanol, a

vehicle control. The results indicated that the AED treatment led to a significant increase

in the survivability when compared to vehicle control. In fact only 12 of the 40 or 30%

of vehicle control-mice survived, while the AED treated mice had 36 of 45 or 80%

survive (Padgett et al. 2000). Thus, this result led to an attempt to discover how this

2 increase in survivability occurred and whether the treatment was having an effect at the cellular level.

Hypothesizing that pharmacological treatment of the host resulted in less severe influenza infection, the focus of this project was to determine if there was a mechanism in which AED, modified the host cell’s interaction with the virus thereby inhibiting viral replication. AED has been shown to enhance the adaptive immune response to influenza virus leading to a higher survival rate of the host. The data also appear to suggest that early events during infection may contribute to the increased survivability of the infected hosts (Padgett et al. 2000). However, the events that occur during the early stage of infection at the primary site of viral replication are only now being discovered.

Like most androgen steroid hormones, AED drives some of its effects through the androgen receptor, and in so doing can have both non-genomic and genomic influences.

In this study, we have attempted to delineate how androstenediol, at concentrations above the physiological concentrations, can influence the replication of the influenza virus. In doing so, knowledge of the mechanism(s) by which AED increases the chance of survival from an influenza virus infection will be generated and may provide important information for the development of new therapeutic strategies for preventing or treating an influenza viral infection.

1.2 Influenza

Influenza, commonly referred to as the flu, is a highly contagious respiratory viral infection. The characteristic signs of an influenza viral infection are high fever, muscle

3 pain, headache, weakness, general discomfort, non-productive cough, sore throat, and runny nose (Monto et al. 2000). According to the Center for Disease Control, in the

Northern hemisphere, a normal influenza season begins in the middle of fall and peaks near the middle of winter. Thus, most transmission occurs during the portion of the year in which temperature decreases and the relative humidity remains low. This observation led to a study that shows the influenza virus remains more stable under cooler conditions

(Lowen et al. 2007). It has also been proposed that lower relative humidity allows ejection of the virus to remain in smaller water droplets, thus allowing it to remain airborne for a longer length of time (Lowen et al. 2007). Thus, one way transmission occurs is from inhaling water droplets containing the influenza virus. Another route of transmission occurs by touching a surface (ex. hand, door knob, etc.), contaminated with water droplets containing influenza virus and then introducing the virus to a respiratory mucosal surface. Although the characteristics of the infection have been noted for thousands of years, understanding how an influenza viral infection could be prevented

(by attempting to inhibit transmission or replication) has only recently been developed as modern molecular techniques have been employed to study the virus.

The influenza virus is believed to have been around since 412 BC (Before Christ), when it was first described by Socrates, and humans have been trying to eliminate it since the first recorded influenza pandemic in 1580 (Ghendon 1994). The first attempt at vaccination was in the mid 1930’s and the results of the experiment were inconclusive, but it was believed that the increase seen in antibodies may have suggested a possible prevention of influenza virus (Francis & Magill 1937). Later, it was shown that vaccines

were able to prevent influenza, although it quickly became apparent that the effectiveness

of an influenza virus vaccine generally decreased as the years past. Thus other treatments

such as antivirals were developed and have also shown decreased efficacy after multiple

applications (Boltz et al 2010). To understand why this occurs one may need to

appreciate the interactions between the influenza virus and host’s response.

Influenza A virus is a negative polarity, single-stranded, segmented RNA virus whose 8 RNA segments encode for 11 proteins including the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), matrix protein (M1), nucleoprotein (NP), polymerase base fragment 2 (PB1-F2), RNA polymerases PB1, PB2, and PA, ion channel

(M2), and nonstructural proteins (NS1 and NS2) (Clifford et al. 2009). The virus gains entry to the body through an airway passage. Then HA on the viral envelope binds to sialic acid-containing receptors on the surface of respiratory epithelial cells and induces clatherin-mediated endocytosis. Neuraminidase cleaves the sialic acid and plays an important part in viral entry and release. HA-specific antibodies prevent either attachment of the virus to the host or intra-endosomal fusion, and if HA-specific antibody levels are high enough they may completely prevent the initiation of infection

(Mozdzanowska et al. 1997). Neuraminidase-specific antibodies are also protective, but unlike HA-specific antibodies, the NA specific antibodies are important in suppressing subsequent viral replication and not the initial entry into cells (Kilbourne et al. 1968,

Murphy et al. 1972, Powers et al. 1996). Current vaccines for influenza virus act by inducing neutralizing antibody (primarily IgA and IgG) responses to specific epitopes of

HA (i.e., neutralizing epitopes: aa92 – 105, 127 – 133 and 183 – 195) and to NA in a

heterotypic fashion (Li et al. 2003).

The upper respiratory tract is initially infected by the virus, and this causes systemic (i.e., fever, headaches and severe malaise) and local respiratory (i.e., nonproductive cough, sore throat, and rhinitis) symptoms. While the incubation period is approximately 1-5 days, the period of infection that has the greatest chance of communicability is within the first 3 days. Respiratory epithelial cells are the primary targets for influenza A virus infection, and these cells serve as the main factories for the production of large numbers of viral progeny (Schmolke & García-Sastre 2010, Shieh et al. 2010, Yu et al. 2011). As the infection progresses, the virus spreads to lower respiratory tissues and if left unchecked can cause progressive primary viral pneumonia or the more common secondary bacterial pneumonia (Rello & Pop-Vicas 2009). In extreme cases or in susceptible individuals (such as the very old and very young), lethal symptoms can occur such as pulmonary edema and acute respiratory distress syndrome, which is associated with massive infiltration of mononuclear leukocytes (Taubenberger &

Morens 2008). This can be associated with haemophagocytosis in which macrophages, presumably over-activated by high levels of inflammatory cytokines, begin to phagocytose and destroy erythrocytes, leukocytes, and platelets (Taubenberger & Morens

2008). As our study will focus specifically on the early innate response from the lung epithelial cell, we will not be providing much detail on the adaptive response or the transition from the innate to adaptive response, but a thorough review of how innate cells are involved in the regulation of the adaptive immune response during influenza a viral

infection can be found in a 2009 review (McGill et al. 2009). Another 2009 article,

details the discovery of other receptors that are essential for the adaptive response to the

influenza virus (Ichinohe et al. 2009) and fits with a short general review of innate

regulation of adaptive immunity (Iwasaki & Medzhitov 2010).

Understanding the human’s response to infection and subsequent recovery should

lead to an important health question. Why doesn’t an infection or vaccination create an

adaptive immune response in which anti-viral antibodies and life-long immunological memory prevent future infections? The reason is based on the fact that the antibodies that are generated in response to the antigens of the virus (or vaccine) only negatively select a very specific subtype. In addition, that negative selection of the antibody- neutralized strains actually positively selects alternative viral subtypes that are not recognized by circulating antibody. In other words, the presence of a specific antibody puts immunological pressure on the mixed influenza virus population and helps facilitate

productive antigenic shift and antigenic drift leading to multiple viral subtypes for which

there is no immunological memory. Antigenic shift is the result of two different strains

mixing their gene segments to form a new infectious subtype, involving animals, such as

poultry or swine (Nelson & Holmes 2007, Treanor 2004). High variability can also be

caused by antigenic drift or the random accumulation of mutations in viral genes coding

for proteins (Nelson & Holmes 2007, Treanor 2004). This high mutation rate is

attributed to a less than efficient viral RNA polymerase combined with the lack of

proofreading during nucleic acid replication. The result of frequent random accumulation

of errors in the RNA leads to amino acid changes, which exposes different antigenic

surfaces. Thus the ability of the virus to change so readily is the major reason why

researchers in the influenza virus field have decided to examine whether modifying the

cell that the virus infects is a better way to combat the influenza virus.

1.3 Cellular Recognition and Reaction to Influenza Viral- Transcriptional control of the innate immune response to influenza virus

At the time a cell becomes infected with influenza virus, the respiratory epithelial cell sends out a call for help; the virus triggers that alarm signal. This occurs because innate immune recognition detects conserved microbial products that are unique to microorganisms. The microbial products that are recognized are typically essential to microbial function and are conserved among closely related species. For example, hemagglutinin and neuraminidase are viral products that are not manufactured by eukaryotic cells. Normally the structures that could recognize these conserved pathogen- associated products would not recognize host components; their presence would thus signal an infection. These conserved targets of innate immune recognition are called pathogen-associated molecular patterns (PAMPs) (Pichlmar & Reis, 2007) and the receptors on or within host cells are called pattern-recognition receptors (PRRs) (Meylan et al. 2006).

PRRs function to induce phagocytosis, activate proinflammatory signaling pathways, activate complement cascades, and induce apoptosis The PRRs include Toll- like receptors (TLRs) and cytoplasmic retinoid acid inducible gene-I (RIG-I)-like receptors (RLRs). In mammals, PRRs that are capable of detecting an infecting pathogen

drive the transcription of genes that activate cells of the innate immune system and

subsequently stimulate the adaptive immune system. Although PRRs were initially

thought to be transmembrane proteins expressed on the surface of the cell, we now know

that several of the PRRs, such as TLR3 (toll like receptor 3) and TLR8 (toll like receptor

8), are localized on the internal surfaces of endosomal/lysosomal compartments (Gorden

et al. 2006). In this configuration, they are more likely to interact with molecular patterns

of viral replication. For example, poly I:C (polyinosine-polycytidylic acid), which mimics viral double-stranded RNA, stimulates TLR3 (Kimura-Takeuchi et al. 1992), whereas imiquimod or resiquimod, which mimic viral single-stranded RNA activate

TLR7 (toll like receptor 7) or TLR8 (Hattermann et al. 2007).

Although stimulation of any of the PRRs results in the activation of a shared group of inflammatory cytokine genes, each PRR is responsible for its own ‘fingerprint’ that drives the immune response towards an anti-viral, anti-bacterial, anti-parasite, or anti-fungal response (Akira et al. 2006, Kawai et al. 2010). Briefly, after interaction with their respective ligands, all TLRs except TLR3 utilize the adapter protein, MyD88

(myeloid differentiation primary response gene (88)), to activate TRAF6 (tumor necrosis factor receptor-associated factor 6) through the IRAK (interleukin receptor-associated kinase) pathway. In contrast, TLR3 uses the adapter protein, TRIF (TIR-domain- containing adapter-inducing interferon-β), in place of MyD88, to activate TRAF6 (Hoebe et al. 2003, Oshiumi et al. 2003, Yamamoto et al. 2003). Regardless, both MyD88 and

TRIF activation lead to NFκB (nuclear factor κB) transcriptional activity. The activation of NFκB results in the transcription of the cytokine genes shared by all members of the

TLR family. Somewhat unique to TLR3 ligation is the induction of IRF3 (interferon

regulatory factor 3), which activates transcription of the anti-viral type I IFNs

(interferons) (Yamamoto et al. 2003). Likewise, TLR8 activation drives type I IFN

expression albeit by utilizing IRF7 (interferon regulatory factor 7) instead of IRF3

(Matsumoto et al. 2004). Thus, although influenza-mediated activation of the TLRs,

drives the expression of the common ‘inflammatory’ genes through NFκB, their specific

activation of TLR3 and TLR8 would selectively drive the expression of a subset of genes

responsible for activation of the appropriate anti-viral immune response (i.e., the type I

interferons). Those TLR3 and TLR8-induced genes would, in part, be dependent on the

transcriptional activity of IRF3 and IRF7. Recognition of the influenza virus by TLRs in some epithelial cells are limited at best, because TLRs are undetectable or only detectable after upregulation by cytokines such as TNFα (tumor necrosis factor alpha) or IFN β

(Tissari et al. 2005). Because of this, RLRs are thought to be the detection methods for the ‘infected’ respiratory epithelial cells (Siren et al. 2006).

There are several RLRs including RNA helicase RIG-I, melanoma differentiation- associated gene-5 (MDA-5), and RNA-dependent protein kinase (PKR) that are known to recognize some form of dsRNA. Although PKR is able to recognize dsRNA, this regulation only occurs after IFN β has activated it (Clemens & Elia 1997, Samuel 2001).

Currently the most likely PRR involved in the early recognition of influenza virus by epithelial cells is RIG-I. At first it was not clear whether MDA5 and RIG-I were redundant, because both were thought to have showed the ability to bind to poly I:C (a synthetic polyinosinic-polycytidylic acid double-stranded RNA or a substitute for

influenza virus activation). Until recently it was not known that RIG-I was unaffected by

this interaction and currently it has been shown that RIG-I, not MDA5, is the essential

RLR in early cellular recognition of the influenza virus (Pichlmar & Reis 2007).

After recognizing the influenza virus, RIG-I initiates antiviral and pro- inflammatory signaling (Le Goffic et al. 2007). The signaling begins with RIG-I via the recruitment domain interacting with the IFN β promoter stimulator 1 (IPS-1), which then leads to the activation of IRFs 3 and 7 through Traf family member-associated NFκB activator (TANK)-binding kinase 1- and Inhibitor of kappa B kinase i-dependent phosphorylation. IPS-1 also activates NFκB via Fas-associated death domain and regulated intramembrane proteolysis-1-dependent pathways. IRFs 3 and 7 coordinately activate IFN β promoter, while NFκB activates pro-inflammatory cytokines. Thus IFN β is one of the major coalescing points of all the currently known PRRs in the epithelial cell.

IFN β, an antiviral cytokine, is one of only a limited number of cytokines and chemokines that have previously been shown to produce a response to a viral infection in epithelilal cells (Gern et al. 2003, Guillot et al. 2005, Julkunen et al. 2001). The secretion of IFN β is one of the initial antiviral events in viral-infected cells and it has been shown to act on specific receptors of neighboring cells to prevent infection by inducing a number of antiviral genes including PKR and ribonuclease (RNAase) L/2’-5’ oligoadenylate synthetase (OAS) (Goodburn et al. 2000, Haller et al. 2007). The importance of this pathway during an influenza viral infection has been confirmed in vivo by IFN β receptor knockout mice (Bergmann et al. 2000, Durbin et al. 1996). PKR, an

IFN β induced, dsRNA-activated cytokine is capable of blocking protein synthesis through its ability to phosphorylate a subunit of eukaryotic initiation factor 2 alpha (eIF-

2α), thus inhibiting viral replication (Saelens et al. 2001). Thus the IFN β gene is a suitable target to study the early signaling that may potentially affect the replication of influenza viruses.

1.4 Influenza Virus Inhibition and Usage of the Cellular Defense

When influenza viruses enter a mammalian cell, it begins an intracellular battle.

As mentioned previously, the cell activates a number of antiviral defenses to combat the virus; the virus, on the other hand, attempts to circumvent these cellular defenses. To counteract the antiviral effects of IFN induction and PKR activation, many eukaryotic viruses, including influenza, have developed strategies to block PKR activity and inhibit the post-transcriptional processing of cellular precursor mRNAs (messenger RNAs)

(Haller & Weber 2007, Krug et al. 2003). The influenza virus also prevents the cell from recognizing viral dsRNA by binding the dsRNA with a viral nonstructural protein, NS1

(Cheng et al. 2009). NS1 proteins sequester and disguise the dsRNA from host dsRNA receptors, like RIG-I and TLR3, therefore hindering the cellular immune defense mechanisms (Min et al. 2006, Newby et al. 2007).

As the influenza infection continues, a switch from host mRNA transcription to genomic viral mRNA occurs, resulting in synthesis of viral proteins (Engelhardt & Fodor

2006). Another function of NS1 is the inhibition of the posttranscriptional processing of cellular antiviral pre-mRNAs by binding cellular proteins that are required for this

process (Nemeroff et al. 1998). At the same time, this does not affect viral mRNAs,

because viral mRNAs are produced by the viral polymerase and not the cellular

polymerase. Thus, many of the host genes upregulated in the presence of the influenza

virus are unable to be translated into proteins, while the virus is still capable of producing

viral mRNA and protein. Beyond the attempts to counteract the defense that the cell has

evolved, influenza uses other cytokines of the cellular defense to facilitate its own

proliferation.

Just as with other cellular defensive signaling, apoptosis and/or the apoptotic process have been shown to be detrimental (Kurokawa et al. 1999) and essential to the replication of the influenza virus (Wurzer et al. 2003, Wurzer et al. 2004). There are at least three ways that apoptosis is regulated in an influenza infected cell: caspase activation and recruitment domain (CARD, type of death domain), several viral proteins, and transcription of apoptotic factors (Ludwig et al. 2006, Mazur et al. 2008, Morris et al.

2005). The apoptotic pathway most relevant to this study is the transcription of apoptotic factors. An example of the transcription of apoptotic factors would be Fas, Fas Ligand, and TRAIL (TNF-related apoptosis inducing ligand), which are expressed in an NFkB- dependent manner and activate the initiator caspases (caspase 8 and 9) (Ludwig et al.

2006). This led us to understand that a successful balance of the apoptotic process is essential for influenza viral propagation. NS1 sequestering viral RNA is an example of the virus attempting to prevent too early of an apoptotic response from the cell, which would result in little to no replication before cell death. An interruption in this delicate balance may result in decreased replication of the influenza virus. Thus, if such a

treatment as AED were able to disrupt the molecular balancing act that the virus uses, it

may result in the limitation of viral replication.

1.5 Androgen Receptor

AED is a naturally occurring hormone in the human body, as it is a metabolite of

dehydroepiandrosterone (DHEA) a steroid produced by the human adrenal cortex. To

understand how AED has an effect on a cell, one must look at the target molecule that

AED interacts with at the cellular level, which is a nuclear receptor. Nuclear receptors

are proteins that are differentiated by the ligands binding them, and the steroid response

element recognition that interacts with the nuclear receptor (Bain et al. 2007). Most

nuclear receptors contain a conserved set of regions consisting of an amino-terminal

domain, a central DNA-binding domain, and a carboxy-terminal ligand-binding domain.

To become more acquainted with how a nuclear receptor functions, we will focus on one

nuclear receptor, the androgen receptor.

In the cytoplasm androgen receptors (ARs) are inactive steroid receptors. The androgen receptors exist in complexes with chaperones, including heat-shock proteins 70

and 90 that hold their ligand binding domain in constitutively open states (Black et al.

2004, Picard et al. 1987, Hager et al. 2000, Picard et al. 1987, Pratt et al. 2004). Once

bound by an androgen, such as AED, androgen receptors are known to form homodimers

thus activating a nongenomic pathway in the cytosol or traveling to the nucleus where the

androgen receptor binds to androgen response elements (AREs) (Claessens et al. 1996,

Rennie et al. 1993, Shaffer et al. 2004, Verrijdt et al. 1999, Verrijdt et al. 2000). The

14 androgen complex (androgen bound to androgen receptor) is known to activate the

PI3K/AKT (serine/threonine protein kinase) pathway in the cytosol without needing to translocate into the nucleus, although most of the known affects appear to result from transcriptional regulation (Baron et al. 2004, Evans 1988, Giguere et al. 1988,

Mangelsdorf et al. 1995).

Androgen binding an androgen receptor induces a conformational change that reveals the nuclear-localization signal overlapping the DNA-binding domain (Jenster et al. 1993, Simental et al. 1991, Zhou et al. 1994). The translocation of the AR into the nucleus occurs once the NLS can be recognized by an import receptor such as importin-α

(Fahrenkrog et al. 2004, Paschal 2002). Inside the nucleus, more than 500 genes are regulated by androgen/androgen receptor complex in epithelial cells, and these genes are also regulated by many other differing functions (Nelson et al. 2002). Occupancy of the

AREs (androgen response elements) by ARs is known to be cyclic, but the precise timing and co-regulator recruitment have not been described in detail (Kang et al. 2002). One problem that arises is that androgen responsive genes, those bound by an AR at the ARE, have shown that most AREs are further than 10 kilobases (kbs) upstream and/or downstream from the target gene’s transcription start site (Bolton et al. 2007).

Furthermore, a significant portion of androgen responsive genes have been shown to be further than 54 kb away from the ARE (Bolton et al. 2007). Another problem, as with many genes, is that the androgen responsive genes may result in secondary genes being affected. Thus, there are many challenges in determining which genes are direct primary targets of the androgen receptor. Something else to consider is that the androgen

15 receptor, like other nuclear receptors, not only acts as a transcription factor, but also has been found to directly or indirectly interact with other proteins found in the cell (Jasavala et al. 2007, Hedman et al. 2006, Honda 2007, Honda et al. 2008, Zhang et al. 2002).

1.6 Interaction of Influenza Viral Subunits and Androgen Receptors

The influenza virus has been shown to interact with many different host cellular proteins, including several cellular proteins that have also been found to interact with subunits of the influenza viral polymerase complex (Deng et al. 2006, Honda et al 2007,

Honda et al. 2008, Momose et al. 2002). The phase in viral replication where these cellular proteins have been found to interact with the virus is the period after the virus escapes the endosome. After exiting the endosome, viral polymerase complex subunits

(PB1, PB2, and PA (polymerase acidic protein)) and nucleoprotein (NP) enters the host cell nucleus, where synthesis of vRNA, positive-strand RNA, or cRNA occurs. During the transport of the influenza viral polymerase complex subunits, one or more of the subunits are known to interact with at least 9 to 11 human cellular proteins (Deng et al.

2006, Honda 2008, Momose et al. 2002). Three of these cellular proteins have been identified: Ebp1 (ErbB3[epidermal receptor tyrosine kinase]-binding protein), Hsp90

(Heat Shock Protein 90), and Ran binding protein 5 (Honda 2007, Honda et al. 2008,

Naito et al. 2007). Even more notable is that two of these identified cellular proteins

(Ebp-1 and Hsp90) have also been shown to interact with the AR (Honda 2007, Honda et al. 2008, Zhang et al. 2002).

Ebp1’s carboxy-terminal proximal end has been shown to interact with the

catalytic region, for RNA polymerization, of PB1 (Honda et al. 2007). Thus, Ebp1 has

been shown to inhibit RNA synthesis of the influenza virus by binding to PB1 (Honda

2007, Honda et al. 2008, Zhang et al. 2002). This carboxy-terminal region of Ebp1, which interacts with PB1, has also been shown to contain a LXXLL motif that is important for mediating interactions with nuclear receptors, including the amino-terminal domain of AR (Zhang et al. 2002). Ebp1 is known to have many other cellular roles including, but not limited to involvement in apoptosis, protein translation, growth regulation, and the ErbB3 signal transduction pathway (Honda et al. 2007). AR is also known to interact with Hsp90, although there is a lack of specifics as to which domains interact. It is known which domains Hsp90 interacts with PB2; Hsp90s N-terminal domain and its middle region (Momose et al. 2002). Recently many more proteins have been discovered that interact with proteins of the influenza virus, in fact many have been shown to interact with subunits of the viral polymerase complex (Konig et al. 2010).

Together these studies show the potential for an interaction between AR and subunits of the influenza virion. Understanding the complexes that both the androgen receptor and influenza virus form may lead to an effective target to inhibit viral replication.

1.7 Hypothesis

Prior results have shown that treatment with AED increases the survival of the host after infection with influenza (Padgett et al. 1997, Padgett et al. 1998, Padgett et al.

1999, Padgett et al. 2000, Padgett et al. 2000b). In addition, it has been shown in our laboratory that AED appears to act through the androgen receptor in RAW 264.7 cells, a mouse macrophage cell line. This along with the knowledge that the AR and proteins of the influenza virus are known to interact with the same cellular proteins led us to our hypothesis that AED treatment would result in decreased replication of the influenza virus in lung epithelial cells. After determining that inhibition of replication may occur

(Fig. 2.1), we developed a central hypothesis that androstenediol inhibits influenza virus replication by modifying the lung epithelial cell’s interaction with the virus. There are three corollary hypotheses that must be evaluated in order to suggest how this may occur.

First, we hypothesize that AED, through the AR, directly influences the virus encoded polymerase and in so doing inhibits viral replication. To determine whether AED is having a direct effect on the influenza viral polymerase complex through its binding to

AR, we will perform binding assays. Our second corollary hypothesizes that AED can enhance the innate immune response in respiratory epithelial cells. In order to determine whether AED can alter the innate immune response we will determine the gene expression of IFN β. Thirdly, we hypothesized that AED modifies other cellular signaling pathways during infection thus affecting influenza viral replication. To investigate this we will use pathway finder arrays to look at multiple pathways to determine whether AED is able to modify these pathways during an influenza infection.

Taken together these studies are aimed at determining if and how AED inhibits the in vitro replication of influenza virus in human lung epithelial cells.

Chapter 2: Androstenediol Suppresses Influenza Virus-Encoded M1 Gene Expression in Human Respiratory Epithelial Cells

2.1 Introduction

The influenza virus apparently was first described by Socrates in 412 BC and

since the first recorded influenza pandemic in 1580, humans have been trying to

eliminate it (Ghendon 1994). The first attempt at influenza virus vaccination was in the mid 1930’s on a small group of human subjects but the tests were unable to determine if the vaccine would prevent influenza; however there was an increase seen in the number of antibodies against the influenza virus suggesting that prevention of influenza-related

disease was possible (Francis & Magill 1937). Shortly thereafter, it was shown that

vaccines were, in fact, able to prevent influenza infection; the influenza vaccine was first

introduced as a licensed product in the United States in 1944. Soon after the

development of these first anti-influenza vaccines it became apparent that the

effectiveness of an influenza virus vaccine decreased continually year after year as the

influenza virus mutated and escaped the protection afforded by the vaccine-induced

antibodies that were highly specific to the original viral strain. In other words, the

mutated progeny virions were not recognized by the specific vaccine-induced antibodies.

As it became clear that development of long-lived protection against influenza virus with a vaccine was going to be elusive, efforts were redirected toward prevention of viral replication. The first antiviral for the influenza virus was developed in 1966 and

was intended to inhibit the release of influenza viral progeny from infected cells.

Currently, antivirals act in two distinct manners: first there are inhibitors of

neuraminidase (NA) and second there are inhibitors of matrix protein 2 (M2). The

current neuraminidase inhibitors include zanamivir and oseltamivir, while the inhibitors

of M2 include amantadine and rimantadine. The neuraminidase inhibitors act as

competitive inhibitors for sialic acid, thus preventing new viral particles from being

released from infected cells. Sialic acid is known to play an important role in human

influenza viral infections by allowing the hemagglutinin to bind to the cell surface before

both the entrance and release of the virion from the cell. The other antivirals act as matrix protein 2 (M2) inhibitors by affecting the influenza virus M2 ion channel. The

M2 also plays a crucial step in the life cycle of the influenza virus as it allows hydrogen ions to pass through the membrane, enter the virion, and result in a dissociation of the viral matrix protein (M1) from the ribonucleoprotein. This results in the uncoating of the virus and exposes the contents of the virion to the cytoplasm of the cell. However, similar to the vaccination strategy against influenza virus, the current anti-virals target the function of highly variable virus-encoded genes and thus can become ineffective. An example of resistance to these antivirals was shown during the 2005 and 2006 influenza seasons when the rates of resistance to M2 inhibitors reach levels greater than 90% in many countries (Bright et al. 2006). Given the poor ability to prevent or treat influenza viral infection a new approach to combating the virus that does not target highly variable virus-encoded genes is required. One possible approach could be through the use of, the androgen hormone androstenediol (AED).

Previous studies had shown that AED was an effective treatment for influenza in

a murine model (Padgett et al. 1997, Padgett et al. 1998, Padgett et al. 1999, Padgett et al.

2000, Padgett et al. 2000b). The hypothesis that AED might be an effective treatment for

influenza viral infection first came from the work of Loria et al., who showed that the

steroid hormone dehydroepiandrosterone (DHEA) regulated host immunity (Loria et al.

1996). Not only was DHEA able to regulate the host immunity, but it also significantly

increased the protective effect against both viral and bacterial infections. This research

was later followed up with the determination that DHEA’s effects occurred through its

metabolite, AED. Early testing of an enteroviral challenge showed that AED was able to

provide better protection than DHEA (Loria & Padgett 1991). Based on this successful

treatment of an enteroviral infection with AED, experiments were designed to investigate

whether AED would have a similar impact during an influenza viral infection.

This led to three individual studies in mice, which when collapsed into a single

data set, revealed that AED treatment significantly increased survival following a lethal challenge with influenza APR8 virus (Padgett et al. 1997). In fact, 80% of the AED treated mice survived compared to only 30% in the control group (Padgett et al. 1997).

In addition, these studies showed AED significantly increased inflammation at day 3 post infection (p.i.) compared to non-AED treated controls (Padgett et al. 1997). This increased inflammation on day 3 p.i., was associated with accumulation of cells in the draining mediastinal lymph node of infected mice (Padgett et al. 1997). This inflammatory infiltrate resolved by day 7 p.i. in AED-treated mice while continuing in the control mice (Padgett et al. 1997). Thus, these data suggest that AED treatment

resulted in an earlier response to the virus and a quicker clearing of the virus from the

lungs when compared to the control mice.

Because AED appeared to act on the early response to the virus, it was decided

that these experiments would target the earliest site of the antiviral response to the influenza virus, and the virus’s target for replication, the epithelial cell. Thus, we proposed that by modifying the epithelial cell, early viral proliferation would be limited thus preventing viral spread. This strategy seemed plausible since the influenza virion is limited by its small coding capacity and thus relies upon the host’s cellular machinery for many stages of its replication cycle.

To gain a better understanding of the cellular pathogenesis associated with the influenza virus, and to determine the immune mechanisms importance for combating the infection at a cellular level, we chose to look at the primary site of infection, the respiratory tract. The respiratory tract contains multiple cell types, but the site where viral proliferation occurs is the alveolar type II epithelial cell (ATIIs). The alveolar type

II epithelial cells have been shown to be a primary target for influenza viral infection

(Wang J et al. 2009). Thus, the cell line A549, that has been characterized as a type II pulmonary epithelial cell line (Foster et al. 1998, Zhang et al. 2005) and has also been shown to contain both α2-3 and α2-6 sialic acid, with high levels of α2-6 sialic acid, was chosen for study (Kumari et al. 2007, Gulati rt al. 2005). Having a cell displaying α2-6

sialic acid is important because human influenza viral strains have been shown to

predominantly attach to this sialic acid in humans (Ibricevic et al. 2006, Thompson et al.

2006). Taken together, these findings guided us to consider the effects of AED on

influenza viral replication in A549 cells. We hypothesized that treatment with AED

would significantly limit viral replication, as shown by M1 gene expression, in the A549

lung epithelial cells.

2.2 Methods

Cell Culture: The A549 (CCL-185™) human epithelial cell line was cultured in Roswell

Park Memorial Institute 1640 (RPMI 1640) media (Invitrogen, Carlsbad, CA) adjusted to

0.01 Molar (M) 4-(2-hydoxyethyl)-1-piperazineethanesulfuonic acid (HEPES), 10-5 M β-

mercaptoethanol (β-Me), 0.075% sodium bicarbonate, 1.5mM L-glutamine, 50 units/ml

(U/ml) penicillin, and 50 µg/ml streptomycin. The RPMI 1640 culture media was supplemented with 10% fetal bovine serum (FBS) (Biocell Laboratories, Inc., Rancho

Dominguez, CA) and cultures were grown in a 37°C incubator in the presence of 5%

CO2 (carbon dioxide).

Experimental Stimulation and Infection: Approximately one day prior to experimental

stimulation, culture supernatants were removed and refreshed with RPMI 1640

supplemented with 10% FBS. The A549 cellular monolayer was then trypsinized with

GIBCO® Trypsin (Invitrogen), collected in conical tubes, and centrifuged. The media

was then removed and freshly made RPMI 1640 was added to resuspend the cell pellet.

A sample of this suspension was diluted with trypan blue stain and the cell number and

viability were calculated using a counting chamber. Cells were then plated in single

culture dishes at approximately 6 x105 cells/ml in RPMI 1640 supplemented with 10%

FBS and allowed to incubate overnight at 37°C incubator in the presence of 5% CO2.

AED (Sigma Aldrich, St. Louis, MO), androstenetriol (AET) (a gift from the Loria

laboratory), dehydroepiandrosterone (DHEA) (Sigma Aldrich), dihydrotestosterone

(DHT) (Sigma Aldrich), flutamide (Flut), hydroxyflutamide (HF), corticosterone (Cort),

and dexamethasone (DEX) (Sigma Aldrich) were prepared fresh before each experiment

at a concentration of 5 x 10-3 M – 1 x 10-4 M solution in a solution of dimethylsulfoxide

(DMSO) and ethanol (ETOH) referred to from herein as DMSO/ETOH (which also served as the vehicle control). The final concentration of DMSO/ETOH in culture media was 0.1% by volume. In all experiments, media was removed and replaced with fresh media containing 10% FBS. Treatment groups were added to stock media preparations at a concentration of 1 µl/ml of 5 x 10-3 M – 1 x 10-4 M solution in a solution of

DMSO/ETOH. Samples that did not receive AED were treated with 1 µl/ml of

DMSO/ETOH or culture media lacking any other solution, as a media control.

Concurrently, treatment groups received media containing 1 hemagglutinatin assay unit

(HAU) of influenza A/PR8/34 (H1N1) virus per ml of culture media.

RNA Isolation: Total RNA isolation was performed by using TRIzol ReagentTM

(Invitrogen) according to the manufacturer’s protocol. The TRIzol method accomplishes this by using a phenol/chloroform based phase separation protocol and is followed by an isopropanol-induced RNA precipitation. RNA precipitates were then washed by adding 1 ml of 75% ETOH. After the RNA wash, the 75% ETOH was decanted and the RNA pellet was resuspended in a range of 20-50 µl of distilled water (dH2O), depending on

pellet size. Total RNA concentrations and purity were determined by a

spectrophotometer. The RNA method was selected on the spectrophotometer to analyze

at 260 nm and 280 nm wavelengths. Concentration of RNA were determined by

measuring the absorbance at 260 nm in a spectrophotometer using quartz cuvettes. Pure

preparations of RNA have an absorbance reading 260/280 value of 1.8 to 2.0. The

values were recorded and used to determine the volume required to equal 1µg of total

RNA. Then the 1µg of total RNA was used as a template to synthesize complimentary

DNA (cDNA) by reverse transcriptase, Promega A3500 reverse transcriptase kit

(Promega, Madison, WI). The manufacturer’s protocol was followed using the random

primer mixtures and then upon completion the final volume was adjusted from 20 µl to

50 µl.

Real Time Polymerase Chain Recation (PCR) analysis of gene expression was accomplished using the TaqMan multiplex method of gene amplification. The final concentration for the PCR reaction was 900 nM for the primers and 100 nM for the probe. The M1 probe was labeled at the 5’ end with the reporter dye 6- carboxyfluorescein (FAM) and at the 3’ end with the quencher dye 6-carboxy-

tetramethyl-rhodamine (TAM). 18S was included in each well and used to standardize

the relative concentrations of cDNA. The 18S probe was labeled at the 5’ end with 2′-

chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) probe and at the 3’ end with

TAM. Samples were prepared by adding 12.5 μl of TaqMan Universal PCR Master Mix

(2X) (Applied Biosystems, Branchburg, NJ) with 2.5 μl of cDNA, 2.5 μl cytokine

25 primer/probe mix, 2.5 μl 18S, ribosomal RNA (rRNA), primer/probe mix, and 5 μl sterile for a total volume of 25 μl. Table 2.1 contains a list of the sequences for primers and probes that were synthesized by Applied Biosystems, Inc. Amplification was performed on the Applied Biosystems ABI Prism 7700 Gene Amplification System (Applied

Biosystems, Foster City, CA) using a two-step process with 40 cycles of 15 second denaturing phase (95°C) and a 1 minute anneal/extension phase (60°C). Sequences for the primer and probe sets for M1 was obtained from van Elden et al. and the sets for 18S rRNA was obtained from Dr. Michael Caligiuri’s (Ohio State University Comprehensive

Cancer Center, Columbus, Ohio).

Quantitation of Gene Expression: Measurement of Real Time PCR data depends on the amount of fluorescent dye detected and the initial amount of amplicon, cDNA template, for amplification. There are excessive quantities of both the amplicon and PCR reagents at the start of the amplification. The detection of the reporter dye early in the Real Time

PCR should follow an exponential growth pattern as a result of each new PCR cycle.

Once the number of amplicons rise and the PCR reagents are exhausted, the rate of increase detection of reporter dye slows into a linear growth pattern. In the final stage the growth pattern reaches a plateau, a point where so much of the reagents have been used that no new reporter dye is detected with the next PCR cycle. Because all samples begin with the same amount of PCR reagents, the point at which they enter into the linear growth phase indicates a similar amount of consumption of the reagents, but will vary based do to the difference in amount of starting amplicon. Then we manually set a

threshold line to intersect the linear growth phase of each graph line and use this to

determine the cycle at which all reactions are at the same relative level of amplification.

The number of cycles that have occurred at this intersection is given the name cycle

threshold or CT. All CT for a particular dye and amplicons were determined from the

same threshold. Once all the CTs were established, there were several methods that could

be used to determine the relative gene expression. We chose to use the comparative CT

method. All the methods have their advantages and disadvantages. The CT method

begins by subtracting the internal control (18S rRNA) from the CT of the experimental

gene (M1 in this case), which results in a ΔCT value for each reaction. ΔCT values

standardize for individual differences in the starting amount of total RNA for each

reaction. The ΔCT values for each of the treatment groups are then averaged and the

averaged ΔCT for the control group is subtracted from the average ΔCT for each test

group. This calibrates datum relative to each experiment’s control group. The resulting

ΔΔCT is then power transformed using the formula 2(-ΔΔCT). This will result in a value of

one for the control group and another number for each treatment group.

Immunocytochemistry: A549 cells were grown on 15-mm coverslips and allowed to

grow for 24 hours before being washed twice with PBS. We treated and infected the cells

simultaneously and allowed the virus to attach to the cells by placing the cells at 4°C for

30 minutes. Then the cells were placed in a 37°C incubator for 30 minutes or 1 hour.

Cells were washed with either acidic PBS (PBS/HCl, 4°C) or PBS (4°C), followed by

two subsequent wash steps with PBS (4°C). Cells were then fixed for 10 minutes with

3.7% formaldehyde (in PBS) at room temperature. After washing, cells were

permeabilised with acetone at -20 C for 5 minutes. Coverslips were then washed with

PBS and blocked with 10% goat serum in PBS for 20 minutes at 37C. All steps beyond this point were performed at room temperature in a humidity chamber. After blocking, cells were incubated with mouse anti-influenza a (Serotec) in PBS for 30 minutes. After

washes with Tween PBS and a single wash with PBS, cells were incubated with an Alexa

fluor goat anti-mouse 488 for 1 hour. Cells were washed three times with Tween PBS and

a single wash with PBS. To visualize cell nuclei and mount cells to slide a DAPI (4',6- diamidino-2-phenylindole) fluorescent mounting medium was used courteously provided by Dr. Yang (Glaser Lab). Fluorescence was visualized using a fluorescence microscope

(Glaser Lab). Analysis of images was performed with ImagePro 6.2 (Fischer Lab).

Statistical Analysis: Statistical analyses were carried out using StatView®. One way or one factor analysis of variance (ANOVA) with M1 ΔCT as a between subjects factor was used to compare differences within studies. Follow up testing; using least significant difference (LSD) post-hoc test was used to make comparisons between treatment groups.

2.3 Results

Influenza Virus M1 gene expression in A549 cells

A549 human lung epithelial cells were treated with AED, DE (vehicle control), or

RPMI/10% FBS (media control) in combination with infection by influenza A/PR9 virus

28 to determine whether or not the steroid had any effect on replication of the virus. Gene expression of the late stage influenza viral protein M1 was used to determine the effect that AED had on influenza viral replication. First a dose curve was performed in order to determine the dosage was optimal for this study (Fig. 2.1). Analysis of these data showed that a concentration of 1 x 10-6 M AED resulted in no significant effect on M1 gene expression. However, a decrease of M1 gene expression was observed at a concentration of 5 x 10-6 M AED and was the amount used in all further studies.

M1 expression was not detected at time points earlier than 6 hours following infection (data not shown). The results at 6 hours p.i. (Fig 2.2), were variable and M1 gene expression was not always detected at this time. Thus, from the time course it was determined that the most appropriate time to further examine M1 expression was 24 hours. The results of this study showed that introducing the virus and treating the epithelial cells with AED at the same time for a period of 24 hours resulted in a significantly decrease in M1 gene expression (greater than 50%), while the vehicular control had no significant affect (Fig.2.3). It was then decided to test whether AED was able to affect viral replication after a cell had become infected. The results showed that treatment of a cell with AED that was already infected with the influenza virus did not result in a decrease in replication as shown by M1 gene expression (data not shown).

After determining that AED was able to decrease influenza viral replication if treatment was given simultaneously with infection, we decided to determine if any other nuclear receptor agonists/antagonists had a similar effect on the viral replication in the A549 cell line.

Comparing different agonists and antagonists of nuclear receptors

The knowledge that the AR is a ligand-dependent transcription factor led us to performed experiments to examine the effect treatment with other steroid hormone nuclear receptor agonists and antagonists. The treatments were comprised of what are mainly known as androgen agonists (AED, AET, DHEA, and DHT), androgen antagonists (CA, Flut, and HF), and glucocorticoid agonists (Cort and DEX). The androgen agonists we chose to use in this experiment were DHEA, of which AED is a metabolite; AET, another DHEA metabolite; and DHT, a well-known potent androgen.

Multiple androgen antagonists, CA, Flut, and HF, were also used in these experiments because all known antagonists of the androgen receptor are recognized to have some agonistic abilities and/or are shown to be metabolized in cells. The glucocorticoids, Cort and DEX, were used because it was previously shown that AED may act to inhibit the effects of glucocorticoids.

The only treatment that indicated a decrease in M1 gene expression was AED, while several of the other treatments appear to indicate an increased level of M1 gene expression (Fig. 2.4). In fact the glucocorticoid agonists, Cort and DEX, showed about a nine-fold increase in M1 gene expression. This large increase in M1 gene expression was not shown in all glucocorticoid agonists as cortisol was tested later and did not show an effect (data not shown). Although AED has been shown in other studies to have an estrogenic effect; estrogen receptor agonists and antagonists were not examined in this study as A549 cells have been shown to lack expression of that specific nuclear receptor.

Thus, these findings indicated that a clear difference between AED and the other tested agonists/antagonists of nuclear receptors. Only AED inhibited M1 gene expression.

Ability of Androstenediol to Inhibit Influenza Viral Entry

To determine whether AED prevented the influenza virus from entering the A549 cells we performed an immunocytochemistry assay. We performed many experiments

(not shown) to determine several different important details: the amount of primary and secondary antibody, the time to allow the influenza virus to enter the cell, type of wash, and blocking agent. In order to perform this experiment we had to allow the influenza virus to attach to the cells, before allowing them a certain time period (30 minutes or 1 hour) to enter. To determine the amount of influenza virus in the cells an acid wash stripped any excess virus from the surface of the cells. Then the virus remaining in the

A549s were cross linked in the cell with formaldehyde and the cells were later permeabilized with acetone to allow the antibodies specific for the influenza virus to enter the cell and bind to the virus. AED treatment did not result in a significant difference when compared to vehicular control (Fig. 2.5 and 2.6).

2.4 Discussion

The study began by determining the best time (Fig. 2.1) and best dosage (Fig. 2.2) of AED to use at the time of infection in the treatment of A549 respiratory epithelial cells. These studies showed that AED decreased gene expression of the late stage influenza viral protein M1 (Fig 2.3). The reason M1 was chosen was because its

expression was shown to be important for the development of a mature influenza virion

and decreased expression of this gene resulted in reduced viral budding of the influenza

virus (Chiang et al. 2008, Gomez et al. 2000, Hui et al. 2004). Bourmakina and Garcia-

Sastre have shown that a threshold amount of M1 protein is essential for the assembly of

viral components into a virion and that release of the virion does not take place until a

sufficient level of M1 protein accumulates at the plasma membrane of a cell (Bourmakina

& Garcia-Sastre 2005). This is unlike other influenza viral proteins such as NA in which

levels can fluctuate with minimal effects on mature infectious virus levels. However,

when M1 protein levels are decreased there is a decrease shown in mature influenza

virions (Bourmakina & Garcia-Sastre 2005). Thus M1 gene expression was chosen as a surrogate for viral replication to study the effects of AED on A549 cells.

Our laboratory had studied AED as an androgen receptor agonist and its abilities to alter immune function both in vivo and in vitro. As discussed earlier in this chapter, other studies have shown AED and other beta-androgens have an ability to inhibit other viral and bacterial infections (Loria & Padgett 1991, Loria et al. 1996). While these studies determined that all the beta-androgens had varying abilities to modify viral or bacterial replication in vitro, we showed that the beta-androgens (DHEA and AET) did not affect influenza viral replication (Fig 2.4). Like the beta-androgens, the other treatment group that was used as an androgen agonist, DHT, did not show a significant modification of the replication of the influenza virus. Androgen antagonists (Flut and

HF) did appear to show a marginal, but insignificant increase in M1, while CA resulted in a nearly three-fold increase in M1 gene expression (Fig. 2.4). Thus adding this to the

AED results suggests that AR may be playing a role in influenza viral replication as

shown by M1 gene expression levels.

The only other treatments to show any modification of M1 levels was the

glucocorticoid agonists (Cort and DEX) with a significant, nearly nine-fold increase in

M1 gene expression (Fig. 2.4). Glucocorticoids have been used in treatment for influenza

viral infection for over fifty years, but more recently there have been warnings issued by

World Health Organization that prolonged use or high dosages of glucocorticoids can result in increased levels of influenza viral replication (Writing Committee of WHO,

2008). This suggests that glucocorticoids, such as dexamethasone, when given to patients who have a lower respiratory infection, may result in an increased viral replication in the respiratory tract. A question that remained was whether the virus was able to enter the cell when treated with AED.

The reason we must look into whether the virus was entering the cell is to determine whether which future experiments should be performed to determine how

AED is modifying the M1 gene replication. There are a couple of different ways found in the literature to answer this question: western blotting of a specific gene such as M1

(Eierhoff 2009) or immunofluorescence imaging with a microscope (Eierhoff 2009,

Karlas 2010). Our group decided to use immunofluorescence imaging with a microscope, which showed (Fig. 2.5 & Fig. 2.6) that AED does not appear to modify the amount of virus entering the A549s.

The experiments described above demonstrate that AED decreased M1 gene expression when AED treatment was given at the time of an influenza viral infection.

Remarkably, AED does not decrease M1 gene expression when it is given post-infection.

AED, also does not appear to prevent the influenza virus from entering A549 cells. This would seem to suggest that the modified cellular or influenza virion has to be treated early on before the influenza virus is able to begin to modify the cellular environment.

Even more surprising was the fact that although androgen antagonist, CA, results in a significant increase, the other androgen hormones do not appear to affect a decrease in

M1 gene expression.

Cytokine Sequence Size Source

M1 Forward 5’-GGACTGCAGCGTAGACGCTT-3’ 189 van Elden

Reverse 5’-CATCCTGTTGTATATGAGGCCCAT-3’

Probe: 5’-CTCAGTTATTCTGCTGGTGCACTTGCCA-3’

18S Forward 5’-CGGTACCACATCCAAGGAA-3’ 187 Caliguiri

Reverse 5’-GCTGGAATTACCGCGGCT-3’

Probe: 5’-TGCTGGCACCAGACTTGCCCTC-3

Table 2.1: Primer and TaqMan Probe Sequences of cytokines M1 and 18S for Real Time

PCR

Dose Curve of AED 1.2

Fold Change) Fold 0.8 -

0.6 * 0.4

0.2

M1 Gene Expression (N M1 Expression Gene CTL DE AED 5x10-6 AED 1x10-6 AED 1x10-7 AED 1X10-8 Treatments (Concentrations in Moles/Liter or M)

Fig. 2.1: Dose Curve of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells. Treatment regimens are represented along the x-axis and the results are reported as fold increase over control cultures. A549 cells (6 x 105 cells/ml) were untreated control (RPMI/FBS), treated with DE (DMSO/ETOH), or varying concentrations (5x10-6 M, 1x10-6 M, 1x10-7 M, 1x10-8 M of AED (in DMSO/ETOH); and infected with influenza virus APR8/34 for 24 hours. M1, a viral gene upregulated late during influenza viral replication, was used to determine results. Treatments were in triplicate. Analysis of gene expression was performed by Real Time PCR. The symbol indicates a significant difference (p<0.05, LSD) between groups.

Influenza Time Course 4.5 4.0 3.5 3.0 2.5 2.0

(M1 Gene Expression) Gene (M1 1.5 1.0 0.5 0.0 6 Hr 6 Hr 6 Hr 12 Hr 12 Hr 12 Hr 24 Hr 24 Hr 24 Hr Infect Ctl Infect DE Infect Infect Ctl Infect DE Infect Infect Ctl Infect DE Infect Fold ChangeFold - AED AED AED N Treatments

Fig. 2.2: Time Course of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells. Treatment regimens are represented along the x-axis and the results are reported as fold increase over control culture at the 6 hour time point. A549 cells (6 x 105 cells/ml) were untreated control (RPMI/FBS), treated with DE (DMSO/ETOH), or AED (5 x 10-6 M in DMSO/ETOH); and infected with influenza APR8/34 virus for varying periods. M1, a viral gene upregulated late during influenza viral replication, was used to assess viral replication. Treatment groups were performed in duplicate. Analysis of gene expression was performed by Real Time PCR.

M1 Gene Expression 1.2

0.8

0.6 * 0.4

0.2 Fofld Change Fofld

- 0

N CTL DE AED Treatment

Figure 2.3: Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells. Treatment regimens are represented along the x-axis and the results are reported as fold-increase over control cultures. A549 cells (6 x 105 cells/ml) were untreated control (RPMI/FBS), treated with DE (DMSO/ETOH), or AED (5 x 10-6 M in DMSO/ETOH); and infected with influenza APR8/34 virus for a period of 24 hours. M1, a viral gene upregulated late during influenza viral replication, levels were decreased by nearly 60%. Analysis of gene expression was performed by Real Time PCR. There were 5 samples per treatment group. The symbol indicates a significant difference (p<0.05, LSD) between groups.

M1 Gene Expression 10 * * 9 8 7 6 5 4 * 3 2 Fold Difference Fold - 1 N 0 DE AED AET DHEA DHT Flut HF CA Cort DEX

Treatment

Figure 2.4: Multiple Steroid Agonists and Antagonists Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells. Treatment regimens are represented along the x-axis and the results are reported as fold increase over DMSO/Ethanol (vehicular control) cultures. A549 cells (6 x 105 cells/ml) were treated with DE (DMSO/ETOH), AED (5 x 10-6 M in DMSO/ETOH) , androstenetriol (AET, 5 x 10-6 M in DMSO/ETOH), dehydroepiandrosterone (DHEA, 5 x 10-6 M in DMSO/ETOH), dihydrotestosterone (DHT, 1 x 10-7 M in DMSO/ETOH), flutamide (Flut, 1 x 10-7 M in DMSO/ETOH), hydroxyflutamide (HF, 1 x 10-7 M in DMSO/ETOH), cyproterone acetate (CA, 1 x 10-7 M in DMSO/ETOH), corticosterone (Cort, 1 x 10-7 M in DMSO/ETOH), and dexamethasone (DEX, 1 x 10-7 M in DMSO/ETOH); and infected with influenza APR8/34 virus for a period of 24 hours. M1, a viral gene upregulated late during influenza viral replication, levels were decreased only with AED treatment. M1 levels were appeared to be increased by the three androgen antagonists (approximately two to three fold) and the two glucocorticoids (Cort and DEX) show a nearly nine fold increase in expression. Analysis of gene expression was performed by Real Time PCR. There were 6 samples per treatment group. The symbol indicates a significant difference (p<0.05, LSD) between groups.

Figure 2.5: The above images were used to determine whether AED had an effect on influenza viral entry. A549s were grown for 24 hrs on cover slips and infected with 10 HAUs of influenza APR/8. Treatment occurred at the same time as infection. Primary antibody (serotec; mouse anti influenza a) used in the experiment was at a concentration of 1:100 and secondary (Alexa Fluor 488 goat anti-mouse) at a concentration of 1:100. In the images above the cells were visualized with a Leica fluorescent microscope using a L5 filter at 40X. Analyses of images were performed using ImagePro 6.2.

Figure 2.6: The above images were used to determine whether AED had an effect on influenza viral entry. A549s were grown for 24 hrs on cover slips and infected with 40 HAUs of influenza APR/8. Treatment occurred at the same time as infection. Primary antibody (serotec; mouse anti influenza a) used in the experiment was at a concentration of 1:100 and secondary (Alexa Fluor 488 goat anti-mouse) at a concentration of 1:100. In the images above the cells were visualized with a Leica fluorescent microscope using a L5 filter at 40X. Analyses of images were performed using ImagePro 6.2.

Chapter 3: Androstenediol’s Effect on Cellular Signaling and a Coalescing of Pattern Recognition Receptors

3.1 Introduction

The goal in the previous chapter was to determine whether androstenediol (AED)

was able to inhibit viral replication at a cellular level: results showed that the influenza

virus matrix protein 1 gene level decreased upon AED treatment. In addition, previous

studies by our lab show that AED contributed to an earlier upregulation of the immune

response to the influenza virus and was also likely clearing the virus from the lungs much

quicker than the control mice. In addition to the general immunological finding,

published studies showed that AED treated mice were able to augment expression of

cytokines of both the innate (i.e., interleukin-1, [IL-1] and tumor necrosis factor alpha,

[TNFα]) and adaptive (i.e., interleukin-2, [IL-2] and interferon gamma, [IFN-γ]) immune responses (Padgett et al. 2000b). In a previous study, AED augmented the expression of

IL-1, IL-2, and TNFα by preventing the glucocorticoid-mediated suppression of these cytokines (Padgett et al. 2000). Thus, the results from the previous chapter showed that

AED treatment limited replication of the virus, next we will determine the mechanism by which replication is limited at the cellular level. The previous studies along with the knowledge that other androgenic hormones tested on epithelial cells have shown regulation of at least 500 genes (DePrimo et al. 2002), led us to examine whether any major pathways or specific genes were modified by AED in epithelial cells. To test

these pathways we decided to again look into respiratory epithelial cells, since they are

the primary target for influenza virus and typically show elevated production of cytokines

in response to influenza viral infection.

Although often shown to produce only a limited number of cytokines, respiratory

epithelial cells are one of the major components in the innate immune response to

influenza viral infection. This response all begins with the recognition of the influenza

virus by the epithelial cell, which then leads to an attempt to prevent replication of the

virus and expression of warning signals to other cells. Respiratory epithelial cells have

intracellular sensors for viral products that once activated begin a signaling cascade that

collectively culminates in the expression of the interferon beta (IFN β) gene (Le Goffic et

al. 2007). It has been shown that retinoic acid-inducible gene-I (RIG-I) is the essential

RIG-I-like receptors (RLR) in early cellular recognition of the influenza virus although

other pattern recognition receptors (PRRs) have been shown to play a role as well

(Rehwinkel & Reis 2010). After recognizing the influenza virus, RIG-I signaling

initiates the antiviral response through the interferon beta promoter and pro-inflammatory signaling through nuclear factor kappa B (NFκB) (Rehwinkel & Reis 2010). All of the known PRRs that have been shown to recognize viruses, including the influenza virus, initiate signaling pathways that coalesce at the activation of interferon regulatory factor-3

(IRF3), interferon regulatory factor-7 (IRF7), and/or NFκB which leads to transcriptional induction of the IFN β gene (Kawai & Akira 2007, Kawai & Akira 2008, Lee & Kim

2007, Takeuchi & Akira 2010).

The effects of IFN β occur through the binding of IFN β to a cellular surface

receptor, interferon-α/β receptor (IFNAR) on both the infected cell and neighboring cells

(Biron 2001, Darnell et al. 1994, Uze et al. 2007). Binding and activation of IFNAR leads to expression of numerous IFN-stimulated genes which act to prevent replication of the influenza virus. These pathways are of such importance that the influenza virus has a

specific gene, nonstructural protein (NS) 1, whose primary function is to bind to and

directly inhibit RIG-I, thus preventing the recognition of the influenza virus and

transcription of IFN β (Gack et al. 2009, Guo et al. 2007, Haye et al. 2009 ). As has been

shown, activation of IFN β is associated with the prevention of influenza viral replication.

Therefore, we decided to test the hypothesis, that AED modified virus-induced signaling

mechanisms and augmented the immediate early antiviral immune response in the

infected cell, by examining IFN β gene expression. Furthermore, we hypothesized that

AED would likely alter viral modifications in cellular cytokines or pathways similar to

results that were augmented in the animal experiments (Padgett et al. 2000b).

3.2 Methods

Cell Culture: The A549 (CCL-185™) human epithelial cell line was cultured in

Rosswell Park Memorial Institute 1640 (RPMI 1640) media (Invitrogen, Carlsbad, CA)

adjusted to 0.01 Molar (M) 4-(2-hydoxyethyl)-1-piperazineethanesulfuonic acid

(HEPES), 10-5 M β-mercaptoethanol (β-Me), 0.075% sodium bicarbonate, 1.5mM L-

44 glutamine, 50 Units/ml (U/ml) penicillin, and 50 µg/ml streptomycin. The RPMI culture media was supplemented with 10% fetal bovine serum (FBS) (Biocell Laboratories, Inc.,

Rancho Dominguez, CA) and cultures were grown in a 37°C incubator in the presence of

5% CO2.

Experimental Stimulation and Infection: Approximately one day prior to experimental stimulation, culture supernatants were removed and refreshed with RPMI 1640 supplemented with 10% FBS. The A549 cellular monolayer was then trypsinized with

GIBCO® Trypsin (Invitrogen), collected in conical tubes, and centrifuged. The media was then removed and freshly made RPMI 1640 was added to resuspend the cell pellet.

A sample of this suspension was diluted with trypan blue stain and the cell number was calculated using a counting chamber. Cells were then plated in single culture dishes at approximately 6 x105 cells/ml in RPMI 1640 supplemented with 10% FBS and allowed to incubate overnight at 37°C incubator in the presence of 5% CO2. AED (Sigma

Aldrich, St. Louis, MO) was prepared fresh before each experiment at a concentration of

5 x 10-3 M solution in a solution of dimethylsulfoxide (DMSO) and ethanol (ETOH) referred to from herein as DMSO/ETOH, also used as the vehicular control. Final concentration of DMSO/ETOH in culture media was 0.1% by volume. In all experiments, media was removed and replaced with fresh media containing 10% FBS.

Samples that did not receive AED were treated with 1 µl/ml of DMSO/ETOH or culture media lacking any other solution, as a media control. Concurrently, treatment groups’

45 media had added 1 hemagglutinating unit (HAU) of Influenza A/PR8/34 (H1N1) virus per ml of culture media.

Focused Microarrays: A GEArrayTM Q Series Kit (Frederick, MD) was used to obtain gene expression in up to 96 cDNA fragments from genes associated with a specific biological pathway. The kit is commercially available and includes RNA preparation; probe synthesis, hybridization, and chemiluminescent detection. Imaging was taken with

GENEGnomeTM a product commercially available through Syngene (Frederick, MD), while GEArray AnalyzerTM performed acquisition and analysis. Membranes were spotted with negative controls (pUC18, etc.) and housekeeping genes (β-actin, GAPDH, etc.). All these products are commercially available through SABiosciences a Qiagen

Company (Frederick, MD).

RNA Isolation: Total RNA isolation was performed after cells were incubated for 4 hr or

24hr by using TRIzol ReagentTM (Invitrogen) according to the manufacturer’s protocol as described in chapter two or Qiagen (Valencia, CA) RNeasy MiniKit. Total RNA concentrations were determined by spectrophotometric readings at 260 and 280 nm wavelengths.

Real Time Polymerase Chain Reaction (PCR) analysis of gene expression was accomplished using the TaqMan multiplex method of gene amplification. The final concentration for the PCR reaction was 900 nM for the primers and 100 nM for the probe. The IFN-beta probe was labeled at the 5’ end with the reporter dye 6- 46 carboxyfluorescein (FAM) and at the 3’ end with the quencher dye 6-carboxy- tetramethyl-rhodamine (TAM). 18S was included in each well and used to standardize the relative concentrations of cDNA. The 18S probe was labeled as shown before.

Samples were prepared by adding 12.5 μl of TaqMan Universal PCR Master Mix (2X)

(Applied Biosystems, Branchburg, NJ) with 2.5 μl of cDNA, 2.5 μl cytokine primer/probe mix, 2.5 μl 18S, ribosomal RNA (rRNA), primer/probe mix, and 5 μl sterile for a total volume of 25 μl. Amplification was performed on the Applied Biosystems ABI

Prism 7700 Gene Amplification System (Applied Biosystems, Foster City, CA) as described previously. Sequences for the primer and probe sets for human IFN-beta was designed using Primer Express software (Applied Biosystems) and the sets for 18S rRNA was obtained from Dr. Michael Caligiuri’s laboratory.

Focused PCR Arrays: A Human Signal Transduction PathwayFinder PCR Array

(Frederick, MD) was used to obtain gene expression in up to 84 key genes representative of 18 different signal transduction pathways. The RT² SYBR® Green qPCR Master

Mixes contained all of the reagents and buffers required for SYBR® Green based real- time polymerase chain reactions in real-time PCR. Each master mix included real-time

PCR buffer, a high-performance HotStart DNA Taq polymerase, nucleotides, and

ROX®. Added the master mix to PCR tubes along with the template and primers were in the microarray plates. Microarray plates have negative controls (pUC18, etc.) and housekeeping genes (β-actin, GAPDH, etc.). All these products are commercially available through SABiosciences a Qiagen Company (Frederick, MD).

Quantitation of Gene Expression: Quantitation was described before with the only two

modifications being the experimental gene being tested and sample size. The CT method

begins by subtracting the internal control (18S rRNA) from the CT of the experimental

gene (IFN-beta in this case), which results in a ΔCT value for each reaction.

Statistical Analysis: Statistical analyses were carried out using StatView®. One way or

one factor analysis of variance (ANOVA) with M1 ΔCT as a between subjects factor was

used to compare differences with studies. Follow up testing, using least significant

difference (LSD) post-hoc test to indicate any significance. Data analysis was performed

on the PCR Array by an integrated web-based software package for the PCR Array

System automatically performs all ΔΔCT based fold-change calculations from the uploaded raw threshold cycle data.

3.3 Results

Interferon Beta Gene Expression in A549 Cells Without Influenza Viral Infection

In the previous experiment our data showed that AED decreased viral replication as shown by M1 gene expression. Thus we now wanted to determine if this decreased expression occurred through the innate immune response of the epithelial cell, thus PRRs.

The gene expression of IFNβ was used as a surrogate to determine the effect that AED had on PRRs, as all known PRRs signaling influenza virus infection are shown to

48 coalesce at the induction of IFN β gene expression. A549 human lung epithelial cells were treated with AED or DE (vehicle control) to determine whether or not the steroid had any effect on IFN β gene expression. This study which allowed the uninfected cells to be treated with AED for 24 hours showed that AED did not significantly alter the gene expression of IFN β as compared to the vehicular control, thus showing no effect (Fig.

3.1).

Focused Microarray in A549 Cells Without Influenza Viral Infection

Knowing that the AED did not affect IFN β on its own without infection we decided to see if it had any effect on the A549 cells in the absence of influenza viral infection. Thus, we again used A549 cells that were treated with AED or DE to determine whether or not the steroid alone had any effect on multiple cellular pathways.

This study allowed the cells to be treated with AED for 4 hours and showed that AED did not significantly alter the expression of any of the 84 genes that were tested, thus again showing no effect of AED alone (Fig. 3.2).

Interferon Beta Gene Expression in A549 Cells With Influenza Viral Infection

Having determined that AED treatment did not have any effect on multiple genes without stimuli, we decided to study IFN beta gene expression with influenza virus infection. A549 human lung epithelial cells were treated with AED or DE to determine

whether or not the steroid had any effect on IFN beta gene expression during an infection

with the influenza virus. The study which allowed the cells to be treated with AED and infected with influenza virus for 24 hours showed that AED did not significantly increase gene expression of IFN β. In fact the infected cells that were treated with AED had a significant decrease in the amount of IFN β gene expression (Fig. 3.3).

PCR Array of A549 Cells With Influenza Viral Infection

Since AED treatment did not affect IFN β gene expression when the cells were infected by influenza virus, we decided to examine whether there was any noticeable effect of AED treatment of influenza viral infection on the multiple cellular pathways.

The study which allowed the epithelial cells to be treated with AED and infected with the influenza virus for 24 hours, showed that AED did significantly increase expression of

22 genes involved in 14 pathways by at least 2.5-fold (Table 3.1). Two major pathways,

NFκB and NFAT (Nuclear Factor of Activated T-cells), showed multiple genes being increased along with a gene or two with expression levels increased by 9-fold or greater

(Fig. 3.4). Also two genes that had previously been shown to have been augmented in the mouse model increased significantly in this array were TNF and IL-2 (Fig. 3.4). Thus these findings indicate that the AED-mediated effect may result from an ability to prevent the suppression of gene induction by the influenza virus.

3.4 Discussion

These studies showed that AED was unable to significantly modify gene

expression of those genes on the PCR array without influenza viral infection, including

that of IFN β gene expression. The effect of AED did not appear to be occurring through

pattern recognition receptors as IFN β gene expression did not show an increase during

influenza viral infection. In fact, it appears to show a significant decrease in IFN β gene

expression. This led us to examine whether AED could be having an effect on other

pathways of the epithelial cell that may result in decreased viral replication.

Thus the AED treated samples that were concurrently infected with influenza

virus indicated several pathways that may be involved in the inhibition of the viral replication and also showed potential for increased adaptive immune signaling. IL-2,

which has been shown to be weakly expressed in A549s (Hurteau et al. 2007), was shown

to have nearly a ten-fold increase in expression when treated with AED in the presence of

influenza virus infection. Thus this is similar to the AED augmented expression of IL-2

shown in the previous animal model (Padgett et al 2000b). The binding of IL-2, a factor

in both the NFκB and NFAT pathways, to the IL-2 receptor results in the differentiation,

growth, and survival in specific T cells (Beadling and Smith 2002, Chow et al. 1999,

Iwashima et. al 2002, Lindemann et al. 2003). Thus this is unlikely to be contributing to

the decreased influenza viral replication at the cellular level for respiratory epithelial cells

in this in vitro model. But there were other genes that were significantly increased in the

array when treated with AED in the presence of influenza viral infection that were also

surrogates of the NFκB and NFAT pathway.

Some of the NFκB pathway surrogates shown to be increased in this array such as nitric oxide synthase 2 (NOS2) and tumor necrosis factor (TNF), have been determined to lead to an increase in nitric oxide (NO) (de Vera et al. 1996, Vila-del Sol et al. 2007).

Although NOS2 and TNF expression are detrimental to the lungs, NO has been shown to be an effective inhibitor of the influenza viral replication (Rimmelzwaan et al. 1999). An increase in NO has been previously shown to correlate with the inhibition of viral RNA synthesis and has been suggested to affect an early step in the replication cycle of the influenza virus (Rimmelzwaan et al. 1999). The other significantly increased pathway,

NFAT pathway, had surrogates increased in this array such as fas ligand and cluster of differentiation 5. These surrogates have been shown to require an accumulation of NFAT for increased expression of their genes, thus suggesting that NFAT has increased expression as well. NFAT factor, NF90, has recently been shown to have a direct effect on influenza viral replication (Wang P et al. 2009). It has been suggested that NF90 decreases early viral replication by targeting the influenza virus nucleoprotein function in the nucleus (Wang P et al. 2009). Thus the increased expression in surrogates of NFAT pathway may lead to a mechanism by which AED contributes to the decreased viral gene expression. Our data, along with data from previous studies (Rimmelzwaan et al. 1999 and Wang P et al. 2009) would suggest that a further examination of NO and NF90 would be beneficial to confirm whether one or both of these products play an essential role in the decreased influenza viral expression we have observed with AED treatment.

Thus, this leads us to question how AED treatment results in the modification of these signaling pathways, and what role the androgen receptor has in these effects.

Figure 3.1: Data of Interferon Beta Gene Expression in A549 Lung Epithelial Cells. Treatment regimens are represented along the x-axis and the results are reported as fold increase over control cultures. A549 cells (6 x 105 cells/ml) were treated with DE (DMSO/ETOH) and AED (5 x 10-6 M in DMSO/ETOH) for a period of 24 hours. IFN- beta is a cellular gene that coalesces all the pattern recognition receptors at a single point. IFN-beta levels showed no significant change in cells that were uninfected. Treatment groups were performed in triplicate. Analysis of gene expression was performed by Real Time PCR.

Figure 3.2: Focused microarray analysis of AED activated genes in A549 cell culture without exposure to influenza virus. A549 cells (6x105cells/ml) were treated with DE (DMSO/ETOH) and AED (5 x 10-6 M in DMSO/ETOH). The microarray has 84 key genes representative of 18 different signal transduction pathways. Up-regulated genes are those with a greater than 2-fold change and down regulated genes are those that are at a 50% decrease. There were also genes on the microarray such as GAPDH to be used as controls. There was no significant change seen between the two treatments.

IFN ß Gene Expression 1.2

0.8

0.6 *

0.4

Fold Difference Fold 0.2 -

N 0 DE AED Treatment

Figure 3.3: Data of Interferon Beta Gene Expression in A549 Lung Epithelial Cells With Exposure to Influenza Virus. Treatment regimens are represented along the x- axis and the results are reported as fold increase over control cultures. A549 cells (6 x 105 cells/ml) were treated with DE (DMSO/ETOH) and AED (5 x 10-6 M in DMSO/ETOH) and infected with influenza virus for a period of 24 hours. IFN-beta is a cellular gene that coalesce all the pattern recognition receptors at a single point. IFN-beta levels have appeared to show a significant decrease in cells that were infected. Analysis of gene expression was performed by Real Time PCR. Treatment groups were performed in triplicate. The symbol indicates a significant difference (p<0.05, LSD) between groups.

Comparing AED gene expression to vehicle control(DMSO:EtOH) Fold Change Genes Pathways Genes Involved In: 11.4716 p450 enzyme aromatase CREB Pathway 10.8528 cluster of differentiation NFAT Pathway 9.6465 interleukin 2 NFkB Pathway NFAT Pathway Calcium and Protein Kinase C Pathway 8.2249 lymphotoxin alpha Insulin Pathway 7.5685 homeobox protein engrailed 1 Hedgehog Pathway and Retinoic Acid Pathway 6.8211 colony stimulating factor 2 Calcium and Protein Kinase C Pathway and LDL Pathway 6.774 kallikrein-related peptidase 2 Androgen Pathway 6.4531 E-selectin LDL Pathway 6.2333 Proto-oncogene protein Wnt-1 Hedgehog Pathway 6.0629 nitric oxide synthase 2 NFkB Pathway Jak-Stat Pathway Phospholipase C Pathway 6.021 matrix metalloproteinase, stromelysin 2 Jak-Stat Pathway 5.9794 fas ligand NFAT Pathway 5.9381 leptin Insulin Pathway 4.5631 Proto-oncogene protein Wnt-2 Hedgehog Pathway 4.2281 platelet/endothelial cell adhesion molecule-1 NFkB Pathway 4.0278 Wnt-1 induced secreted protein 1 Wnt Pathway 3.5064 hexokinase 2 Insulin Pathway 3.1167 selectin P ligand LDL Pathway 3.0525 vascular cell adhesion molecule-1 NFkB Pathway Phospholipase C Pathway LDL Pathway 2.9897 interleukin 4 Jak-Stat Pathway 2.9282 cyclin-dependent kinase inhibitor 2B TGF-ß Pathway 2.9079 tumor necrosis factor NFkB Pathway

Table 3.1: PCR array analysis of AED activated genes in A549 cell culture with exposure to influenza virus. A549 cells (6x105cells/ml) were treated with DE (DMSO/ETOH) and AED (5 x 10-6 M in DMSO/ETOH) with infection of influenza virus for a period of 24 hours. The PCR array has 84 key genes representative of 18 different signal transduction pathways. Up-regulated genes are those with a greater than 2.5-fold change and down regulated genes are those that are at a 67% decrease. There were also genes in the PCR array such as GAPDH to be used as controls. There were 22 genes involved in 14 pathways that were upregulated in the AED treated PCR array as compared to the DMSO:EtOH.

Chapter 4: Determining Whether Androstenediol Modifies Influenza Virus-Encoded M1 Gene Expression Through the Androgen Receptor

4.1 Introduction

Nuclear receptors (NRs) vary in their ability to bind hormones and in their functions once hormones are bound to the intracellular receptor. The primary function of a nuclear receptor is to activate genes by acting as a ligand-activated transcription factor

(Robinson-Rechavi et al. 2003), although another postulated function of nuclear receptors is to act through non-genomic signaling (Foradori et al. 2008). Characterized by their conserved DNA-binding domains, NRs are divided into at least four classes by dimerization patterns and response elements (Denayer et al. 2010, Germain et al. 2006,

Glass 1994). Two of these classes contain known receptors for the ligand, androstenediol

(AED): androgen receptor (AR) and estrogen receptor. Interestingly, the lung epithelial cell line (A549) used in our studies has been shown to lack expression of the estrogen receptor. However, the AR has not only been shown to be expressed in the A549 cell line (Provost et al. 2000), but the AR has previously been demonstrated in our lab to bind to a promoter complex in another cell line upon treatment of AED (Farrow 2007). Thus, our studies focused on the androgen receptor as our lab has previously investigated and has seen effects of AED through the activation of the androgen receptor.

Thus if AED is acting through the AR in A549s it could be effecting the virus by modifying cellular genes or physically interacting with other proteins that may have an effect on influenza viral replication. The modifying of cellular genes or transcriptional 57

effect of androgens have been shown to be the result of a direct binding of the androgen

receptor dimer to an androgen response elements on genes (Wong et al. 1993). In fact a recent study suggested that nearly two hundred genes have been shown to be modified in

A549s upon treatment with testosterone (Mikkonen et al. 2010). Another way AED may be resulting in the modification of influenza viral replication would be through AED bound AR physically interacting with proteins that have been shown to interact with subunits of the influenza virus. In fact two cellular proteins (Ebp-1 and Hsp90) that have

been shown to interact with subunits of the influenza virus polymerase have also been

shown to interact with the AR (Honda 2008, Honda et al. 2007, Zhang et al. 2002).

Ebp1, which is known to physically interact with the AR, has been shown to inhibit RNA

synthesis of the influenza virus by binding to PB1 (Honda 2008, Honda et al. 2007,

Zhang et al. 2002). HSP90, is the other protein known to interact with the AR and bind

to the influenza viral protein PB2 (Momose et al. 2002). Other interactions with the AR

have been shown as well, including the effects of AR in cells treated with AED.

In fact previous studies from our laboratory have shown that AED-induced activation of the AR is able to alter the effect of the activated glucocorticoid receptor

(GR). This affect has been shown to modify the effects of activated glucocorticoid transcription activation, gene expression patterns during wound healing, and the ability to ameliorate the suppressive effects on the gene expression of TNF-α (Farow 2007, Head et al. 2006). The activated-GR has been shown to antagonize pro-inflammatory gene expression. The mechanism by which activated-GR suppresses pro-inflammatory signaling pathways is through the interaction/inhibition of the function of both AP1 and

NFκB (De Bosscher et al. 2000, De Bosscher et al. 2003). This information combined

with our previous observations that indicated an increase in expression of multiple genes

of the NFκB pathway upon AED-treatment of influenza virus infected cells (Table 3.1) and an increase in M1 gene expression (Fig. 2.3) upon treatment with glucocorticoids led us to our hypothesis. We hypothesized that the activated GR effects on influenza viral replication would be abrogated with treatment of AED. Specifically, we hypothesized that the AED-treatment observations of decreased influenza viral replication would be the result of the AED-bound androgen receptor.

4.2 Methods

Cell Culture: The A549 (CCL-185™) human epithelial cell line was cultured in Roswell

Park Memorial Institute 1640 (RPMI 1640) media (Invitrogen, Carlsbad, CA) adjusted to

0.01 Molar (M) 4-(2-hydoxyethyl)-1-piperazineethanesulfuonic acid (HEPES), 10-5 M β- mercaptoethanol (β-Me), 0.075% sodium bicarbonate, 1.5mM L-glutamine, 50 Units/ml

(U/ml) penicillin, and 50 µg/ml streptomycin. The RPMI culture media was supplemented with 10% fetal bovine serum (FBS) (Biocell Laboratories, Inc., Rancho

Dominguez, CA) and cultures were grown in a 37°C incubator in the presence of 5%

CO2.

GIBCO® Trypsin (Invitrogen), collected in conical tubes, and centrifuged. The media

was then removed and freshly made RPMI 1640 was added to resuspend the cell pellet.

A sample of this suspension was diluted with trypan blue stain and the cell number was

calculated using a counting chamber. Cells were then plated in single culture dishes at

approximately 6 x105 cells/ml in RPMI 1640 supplemented with 10% FBS and allowed

to incubate overnight at 37°C incubator in the presence of 5% CO2. AED (Sigma

Aldrich, St. Louis, MO), dihydrotestosterone (DHT) (Sigma Aldrich), cyproterone

acetate (CA) (Sigma Aldrich), dexamethasone (DEX) (Sigma Aldrich), and R1881

(Methyltrienolone) (Sima Aldrich) was prepared fresh before each experiment at a

concentration of 5 x 10-3 M – 1 x 10-4 M solution in a solution of dimethylsulfoxide

(DMSO) and ethanol (ETOH) referred to from herein as DMSO/ETOH, also used as the

vehicular control. Final concentration of DMSO/ETOH in culture media was 0.1% by

volume. In all experiments, media was removed and replaced with fresh media containing 10% FBS. Treatment groups were added to stock media preparations at a concentration of 1 µl/ml of 5 x 10-3 M – 1 x 10-4 M solution in a solution of

DMSO/ETOH. Samples that did not receive AED were treated with 1 µl/ml of

DMSO/ETOH or culture media lacking any other solution, as a media control.

Concurrently, treatment groups’ media had 1 hemagglutinating unit (HAU) of Influenza

Virus A/PR8/34 added per ml of culture media.

RNA Isolation: Total RNA was isolation was performed by using TRIzol ReagentTM

(Invitrogen) according to the manufacturer’s protocol. The TRIzol method accomplishes

this by using a phenol/chloroform based phase separation protocol and is followed by an

isopropanol-induced RNA precipitation. RNA precipitates were then washed by adding 1 ml of 75% ETOH. After the RNA wash, the 75% ETOH was decanted and the RNA pellet was resuspended in a range of 20-50 µl of distilled water (dH2O), depending on pellet size. Total RNA concentrations and purity were determined by a spectrophotometer. The RNA method was selected on the spectrophotometer to analyze at 260 nm and 280 nm wavelengths. Concentration of RNA should be determined by measuring the absorbance at 260 nm in a spectrophotometer using quartz cuvettes. Pure

preparations of RNA have an absorbance reading 260/280 value of 1.8 to 2.0. To make

sure the readings are significant the readings of the 260 nm should be between 0.15 and

1.0. The numbers were recorded and placed in Microsoft® Excel® file to determine the

volume required to equal 1µg of total RNA. Then the 1µg of total was used as a template

to synthesize complimentary DNA (cDNA) by reverse transcriptase, Promega A3500

reverse transcriptase kit (Promega, Madison, WI). The manufacturer’s protocol was

followed using the random primer mixtures and then upon completion the final volume

was adjusted from 20 µl to 50 µl.

Real Time Polymerase Chain Reaction (PCR) analysis of gene expression was

accomplished using the TaqMan multiplex method of gene amplification. The final

concentration for the PCR reaction was 900 nM for the primers and 100 nM for the

probe. The M1 probe was labeled at the 5’ end with the reporter dye 6-

carboxyfluorescein (FAM) and at the 3’ end with the quencher dye 6-carboxy-

tetramethyl-rhodamine (TAM). 18S was included in each well and used to standardize

61 the relative concentrations of cDNA. The 18S probe was labeled at the 5’ end with 2′- chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) probe and at the 3’ end with

TAM. Samples were prepared by adding 12.5 μl of TaqMan Universal PCR Master Mix

(2X) (Applied Biosystems, Branchburg, NJ) with 2.5 μl of cDNA, 2.5 μl cytokine primer/probe mix, 2.5 μl 18S, ribosomal RNA (rRNA), primer/probe mix, and 5 μl sterile for a total volume of 25 μl. Table 2.1 contains a list of the sequences for primers and probes that were synthesized by Applied Biosystems, Inc. Amplification was performed on the Applied Biosystems ABI Prism 7700 Gene Amplification System (Applied

Quantitation of Gene Expression: Measurement of Real Time PCR data depends on the amount of fluorescent dye detection and the initial amount of amplicon, cDNA template, for amplification. There are excessive quantities of both the amplicon and PCR reagents at the start of the amplification. The detection of the reporter dye early in the Real Time

PCR should follow an exponential growth pattern as a result of each new PCR cycle.

with the same amount of PCR reagents, the point at which they enter into the linear

growth phase indicates a similar amount of consumption of the reagents, but will vary

based do to the difference in amount of starting amplicon. Then we manually set a

threshold line to intersect the linear growth phase of each graph line and use this to

determine the cycle at which all reactions are at the same relative level of amplification.

The number of cycles that have occurred at this intersection is given the name cycle

threshold or CT. All CT for a particular dye and amplicons are determined from the same

threshold. Once all the CTs are established, there are several methods that could be used

to determine the relative gene expression, but our lab has chosen to use the comparative

CT method. All the methods have their advantages and disadvantages. The CT method