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Theses and Dissertations (ETD) College of Graduate Health Sciences

12-2013

Determinants of Upper Genital Tract Complications in a Determinants of Upper Genital Tract Complications in a

Chlamydial Urogenital Mouse Model Chlamydial Urogenital Mouse Model

Enitra N. Jones University of Tennessee Health Science Center

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Recommended Citation Recommended Citation Jones, Enitra N. , "Determinants of Upper Genital Tract Complications in a Chlamydial Urogenital Mouse Model" (2013). Theses and Dissertations (ETD). Paper 347. http://dx.doi.org/10.21007/etd.cghs.2013.0156.

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Determinants of Upper Genital Tract Complications in a Chlamydial Urogenital Determinants of Upper Genital Tract Complications in a Chlamydial Urogenital Mouse Model Mouse Model

Abstract Abstract Genital Chlamydia trachomatis infection is a major public health concern. Chlamydia is the most commonly reported infection in the United States and the most common bacterial sexually transmitted infection worldwide. Unrecognized infection endangers female reproductive health by serious complications such as Pelvic Inflammatory Disease, ectopic pregnancy, and involuntary infertility. Widespread Chlamydia control programs were implemented more than two decades ago to improve women's reproductive health but, despite initial success, the number of chlamydial infections reported have increased.

One of the hypotheses put forth to explain increased chlamydial reporting suggests that a longterm caveat of control initiatives is interference with the development of natural occurring immunity as a result of mass screening and rapid treatment. It is proposed that human cohorts are more susceptible to subsequent chlamydiae infection and their increased susceptibility drive the current increase in sexually transmitted chlamydiae case notifications.

In these studies we describe a comprehensive approach to assessing the role of early antichlamydial intervention, an integral component of control initiatives, on the subsequent development and severity of upper genital tract sequelae in a murine model of recurrent chlamydiae urogenital infection. The development of an in vivo model of urogenital Chlamydia trachomatis infection is central to defining the risk of developing long term reproductive complications, delineating potential biomarkers for chlamydial-induced genital tract disease, interrogating host factors that may contribute to the development of adverse complications, and anti-chlamydial vaccine development.

Document Type Document Type Dissertation

Degree Name Degree Name Doctor of Philosophy (PhD)

Program Program Biomedical Sciences

Research Advisor Research Advisor Gerald I. Byrne, Ph.D.

Keywords Keywords Chlamydia, Disease, Genital Tract, Mouse, Sequelae

Subject Categories Subject Categories Medical Sciences | Medicine and Health Sciences

Comments Comments One year embargo expired December 2014

This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/347

Determinants of Upper Genital Tract Complications in a Chlamydial Urogenital

Mouse Model

A Dissertation

Presented for

The Graduate Studies Council

The University of Tennessee

Health Science Center

In Partial Fulfillment

Of the Requirements for the Degree

Doctor of Philosophy

From The University of Tennessee

By

Enitra N. Jones

December 2013

ii

Copyright © 2013 by Enitra N. Jones.

All rights reserved.

iii

DEDICATION

This work is lovingly dedicated to my mother, Evelyn T. Jones. You believed in

me when I was filled with self-doubt. You encouraged me when others said I could not

accomplish the goal. Your faith never wavered and you fervently prayed. For this and

more I am eternally grateful.

iv

ACKNOWLEDGEMENTS

It takes a village . . . .

–African Proverb

First, I would like to thank my research advisor, Dr. Gerald I. Byrne, for the

opportunity to carry out the enclosed research in his laboratory. His passion for science

and enthusiasm for learning have been both inspiring and motivational throughout my

doctoral journey. Dr. Byrne has also allowed me the freedom to integrate my interest in

public health with my academic pursuits and for that I am grateful.

The guidance offered by each member of my graduate committee was essential. I

would like to thank Drs. Robert Belland, B. Keith English, Elizabeth Fitzpatrick, and P.

David Rogers for providing scientific insight throughout this process and for being

genuinely invested in my success while matriculating at the University of Tennessee

Health Science Center and beyond. Whether it was help with a laboratory technique, a

recommendation letter, or an encouraging word, each member was more than willing to

lend a helping hand. Thank you for sharing your expertise and offering thoughtful

critiques on my work.

I would also like to acknowledge past and present members of the Byrne

Laboratory. My first teachers in the laboratory were Drs. O. Sadia Mahdi and Isao

Miyairi who patiently taught me the “ins and outs” of cell culture and chlamydial growth.

I am thankful for Vijaya Onguri’s contributions to early vaccination studies and her

ongoing friendship. The technical help throughout the years provided by Dr. Jan Peters

and Jonathan Laxton, now members of the RBL staff, is greatly appreciated. The

histological studies discussed in this dissertation would not have been possible without

Xeofei Wang. Conversations with Dr. Yin Su concerning animal studies were invaluable.

I also want to acknowledge the RBL staff and the many summer students that have been

active participants in laboratory discussions throughout the years. Thank you all!

My time at the University of Tennessee Health Science Center has been enriched

by several members of the faculty/staff and student body. I was truly blessed to have

made the acquaintance of my “UT Mom”, Mrs. Ruby McNeal. Your unwavering support

and encouragement will never be forgotten and is appreciated beyond words. Thank you

to Mrs. Devonia Cage, Mrs. Carolyn Fields, and Mrs. Evelyn Lewis for your prayer and

encouragement. The guidance and mentorship offered by Executive Vice Chancellor and

Chief Operations Officer, Dr. Kennard Brown, was invaluable and greatly appreciated. I

also want to thank the Microbiology, Immunology, and Biochemistry Department, the

members of the Black Graduate Student Association, and many of my fellow graduate

students for their help throughout the years.

I would like to thank my immediate and extended family for their love and

support. To my parents, Ezra and Evelyn Jones, who prayed without ceasing, dried my

tears when things seemed too difficult to continue, and continue to love me

v

unconditionally. I love you to life forever. To my grandmother, Annie Sue Gibbs, thank

you for reminding me that God doesn’t always “move the mountains”, sometimes He

gives us the strength to climb them! To my aunts, uncles, siblings, cousins, friends, and

sorority sisters- thank you for the comedic relief and unwavering support. To my best

friends (outside of my parents), Tron Foster, Jason Hughes and Luviska Nicholas, thank

you for nourishing my spirit and believing in my dreams. Sincere thanks to two of my

ongoing mentors and friends, Drs. Oswald D’Auvergne and Ivory Toldson. Last but

certainly not least, I thank the Lord for granting me the serenity to accept the things I can

not change, the courage to change the things I can, and the wisdom to know the

difference.

vi

ABSTRACT

Genital Chlamydia trachomatis infection is a major public health concern.

Chlamydia is the most commonly reported infection in the United States and the most

common bacterial sexually transmitted infection worldwide. Unrecognized infection

endangers female reproductive health by serious complications such as Pelvic

Inflammatory Disease, ectopic pregnancy, and involuntary infertility. Widespread

Chlamydia control programs were implemented more than two decades ago to improve

women’s reproductive health but, despite initial success, the number of chlamydial

infections reported have increased.

One of the hypotheses put forth to explain increased chlamydial reporting

suggests that a long-term caveat of control initiatives is interference with the

development of natural occurring immunity as a result of mass screening and rapid

treatment. It is proposed that human cohorts are more susceptible to subsequent

chlamydiae infection and their increased susceptibility drive the current increase in

sexually transmitted chlamydiae case notifications.

In these studies we describe a comprehensive approach to assessing the role of

early anti-chlamydial intervention, an integral component of control initiatives, on the

subsequent development and severity of upper genital tract sequelae in a murine model of

recurrent chlamydiae urogenital infection. The development of an in vivo model of

urogenital Chlamydia trachomatis infection is central to defining the risk of developing

long term reproductive complications, delineating potential biomarkers for chlamydial-

induced genital tract disease, interrogating host factors that may contribute to the

development of adverse complications, and anti-chlamydial vaccine development.

vii

TABLE OF CONTENTS

CHAPTER 1. CHLAMYDIAE .........................................................................................1

Historical Perspective ....................................................................................................1

Taxonomy ......................................................................................................................1 Developmental Cycle .....................................................................................................2 Chlamydiae Clinical Significance ..................................................................................4

Chlamydia pneumoniae .............................................................................................4 Chlamydia psittaci .....................................................................................................4

Chlamydia trachomatis ..............................................................................................6 Ocular Chlamydia trachomatis ..................................................................................6

Genital Chlamydia trachomatis .................................................................................6 Mouse Model of Genital Infection .................................................................................7

Murine Genital Tract Pathology ................................................................................9 Murine Model Limitations .........................................................................................9

Immunological Response to Genital Infection ............................................................11 Intracellular Immune Response Overview...................................................................11

Chlamydia Innate Response .....................................................................................11 Chlamydia Adaptive Response ................................................................................12 Vaccination ..............................................................................................................13

Control Measures .........................................................................................................14 Sexually Transmitted Chlamydia Epidemiology .....................................................15

Hypotheses for Rebounding Chlamydia Rates ........................................................16

Arrested Immunity Hypothesis ................................................................................18

Dissertation Rationale ..................................................................................................19

CHAPTER 2. ARRESTED IMMUNITY: IMPACT OF EARLY TREATMENT

ON UPPER GENITAL TRACT SEQUELAE ..............................................................20

Introduction ..................................................................................................................20

Materials and Methods .................................................................................................21 Mice .........................................................................................................................21 Chlamydia Strain and Titration ................................................................................21 Mouse Infection .......................................................................................................21 Antibiotic Treatment ................................................................................................21

Bacterial Shedding ...................................................................................................21

Fluorescence-Linked Immunosorbent Assay (FLISA) Antibody Analysis .............22

Pathology .................................................................................................................22 Organ-Total Body Weight Ratio Analysis ...............................................................22 Statistical Analysis ...................................................................................................23

Results ..........................................................................................................................23 Kinetics of Genital Tract Infection in Antibiotic-Treated Mice ..............................23

Immune Arrest and Pathology Following Primary Infection of Antibiotic-

Treated Mice ............................................................................................................23 Disease Severity Following Reinfection in Antibiotic-Treated Mice .....................26

viii

IL-4 Is Detectable Late Following Reinfection and Positively Correlates with

Disease Severity in Antibiotic-Treated Mice ...........................................................31 Chlamydia and Cellular Infiltrates Following Reinfection in Antibiotic-Treated

Mice ..........................................................................................................................31

Discussion ....................................................................................................................36

CHAPTER 3. CHLAMYDIAL GENITAL TRACT SEQUELAE IS AGE AND

STRAIN-DEPENDENT ..................................................................................................42

Introduction ..................................................................................................................42 Materials and Methods .................................................................................................42

Mice .........................................................................................................................42 Chlamydia Strain .....................................................................................................43

Chlamydia muridarum Infection .............................................................................43 Pathology Assessment .............................................................................................43

Results ..........................................................................................................................44 C57BL/6J Exhibit Age-Dependent Upper Genital Tract Sequelae Post-Primary

Intravaginal Infection ...............................................................................................44 Secondary Intravaginal Infection Exacerbates Chlamydial-Induced Upper

Genital Tract Complications ....................................................................................44 C57BL/6 Mice Are More Susceptible to Chlamydial-Induced Upper Genital

Tract Complications When Compared to DBA/2J Mice .........................................44

Discussion ....................................................................................................................47

CHAPTER 4. IMMUNIZATION WITH C. MURIDARUM OUTER

MEMBRANE COMPLEX FAILS TO PROTECT AGAINST UPPER

GENITAL TRACT COMPLICATIONS IN A MURINE MODEL OF

GENITAL INFECTION .................................................................................................50

Introduction ..................................................................................................................50

Materials and Methods (Systemic Lethal Model) ........................................................51 Mice .........................................................................................................................51

Chlamydiae ..............................................................................................................51 COMC Extraction ....................................................................................................51 Immunization and Infection .....................................................................................52

Results (Systemic Lethal Model) .................................................................................52 Materials and Methods (Urogenital Model) ................................................................55

Mice .........................................................................................................................55

Chlamydiae ..............................................................................................................55

COMC Extraction ....................................................................................................55 Immunization and Infection .....................................................................................55

Results (Urogenital Model) ..........................................................................................56 Materials and Methods (Alum Study)..........................................................................56

Mice .........................................................................................................................56

Chlamydiae ..............................................................................................................56 Immunization and Infection .....................................................................................59 Pathology Assessment .............................................................................................59

ix

Results (Alum Study) ...................................................................................................59

Immunogens without Intravaginal Chlamydia muridarum Challenge Do Not

Induce Severe Upper Genital Tract Sequelae Formation .........................................59 Alum Alone Does Not Negate the Development of Severe Upper Genital Tract

Complications ...........................................................................................................63 Discussion ....................................................................................................................63

CHAPTER 5. CONCLUSIONS ......................................................................................68

Testing the IL-4 Hypothesis.........................................................................................68 Identifying Genetic Link to Age-Dependent Disease Severity....................................68

Implications for Vaccine Development .......................................................................69

LIST OF REFERENCES ............................................................................................... 70

APPENDIX A. MORBIDITY AND PATHOLOGY SEVERITY POST-

PRIMARY INFECTION IN DEFERRED TREATMENT STUDIES .......................91

APPENDIX B. MORBIDITY AND PATHOLOGY SEVERITY POST-

SECONDARY INFECTION IN DEFERRED TREATMENT STUDIES .................92

APPENDIX C. TH1-RELATED CYOKINES POST RECURRENT

INFECTION IN ARRESTED IMMUNITY MODEL..................................................93

APPENDIX D. TH2-RELATED CYTOKINES POST RECURRENT

INFECTION IN ARRESTED IMMUNITY MODEL..................................................94

VITA................................................................................................................................. 95

x

LIST OF TABLES

Table 1-1. Chlamydiae that cause human disease. ...........................................................5

Table 1-2. Increased chlamydial rate hypotheses. ..........................................................17

xi

LIST OF FIGURES

Figure 1-1. Chlamydiae developmental cycle. ..................................................................3

Figure 2-1. Shedding of viable C. muridarum following intravaginal challenge. ...........24

Figure 2-2. C57BL/6 pathology severity scores 49 days post-primary C. muridarum

intravaginal infection. ..................................................................................25

Figure 2-3. Total Immunoglobulin G (IgG) antibody response 49 days post-primary

C. muridarum intravaginal infection. ...........................................................27

Figure 2-4. Immunoglobulin G (IgG) isotype responses 49 days post C. muridarum

intravaginal infection in mice intraperitoneally treated with doxycycline...28

Figure 2-5. Incidence and severity of upper genital tract sequelae in antibiotic-

treated mice 28 days post-secondary infection. ...........................................29

Figure 2-6. Incidence and severity of uterine horn and oviduct sequelae in antibiotic-

treated mice 28 days post-secondary infection. ...........................................30

Figure 2-7. Weight ratio verses total severity score correlative analysis. .......................32

Figure 2-8. Comparative analysis of weight ratios post-primary and secondary

sacrifice in untreated intravaginally infected C57BL/6 mice. .....................33

Figure 2-9. Systemic IL-4 post recurrent infection. ........................................................34

Figure 2-10. Neutrophil infiltrates post-secondary infection. ...........................................35

Figure 2-11. Mast cell infiltrate post-secondary infection.................................................37

Figure 2-12. Mast cell infiltrate verses systemic IL-4 correlative analysis. ......................38

Figure 2-13. Eosinophil infiltrate post-secondary infection. .............................................39

Figure 3-1. Upper genital tract disease incidence and severity post-primary

infection........................................................................................................45

Figure 3-2. Upper genital tract disease incidence and severity post-secondary

infection........................................................................................................46

Figure 3-3. Weight ratio disease severity validation post-primary and secondary

intravaginal infection. ..................................................................................48

Figure 4-1. High dose 6BC C. psittaci systemic infection survival curve. .....................53

Figure 4-2. Low dose 6BC C. psittaci systemic infection survival curve. ......................54

xii

Figure 4-3. Comassie stained SDS-Page gel of C. muridarum (MoPn)-derived

COMC and C. psittaci (6BC)-derived COMC preparations. .......................57

Figure 4-4. C. muridarum-derived COMC immunization study gross pathology. .........58

Figure 4-5. Alum study experimental timeline. ...............................................................60

Figure 4-6. Oviduct pathology severity of non-challenged immunized groups. .............61

Figure 4-7. Uterine horn pathology severity of immunized, non-challenged groups. .....62

Figure 4-8. Oviduct pathology severity of immunized and challenged groups. ..............64

Figure 4-9. Uterine horn pathology severity of immunized and challenged groups. ......65

xiii

LIST OF ABBREVIATIONS

APC Antigen-presenting cells

CDC Centers for Disease Control and Prevention

Cpn Chlamydia pneumoniae

Ct Chlamydia trachomatis

DC Dendritic cell

EB Elementary bodies

ELISA Enzyme-linked immunosorbent assay

FLISA Fluorescent-linked immunosorbent assay

GM-CSF Granulocyte-macrophage coloy-stimulating factor

H&E Hematoxylin and eosin stain

HPV Human papillomavirus

HSP Heat shock protein

IACUC Institutional Animal Care and Use Committee

IFN Interferon

IFU Inclusion-forming units

IgG Immunoglobulin G

IL Interleukin

LD50 Lethal dose, 50%

LGV Lymphogranuloma venereum

LPS Lipopolysaccharide

MHC Major histocompatibility complex

MOMP Major outer membrane protein

MoPn Chlamydia muridarum

NAAT Nucleic acid amplification testing

PAMP Pathogen-associated molecular pattern

PID Pelvic inflammatory disease

PMN Polymorphonuclear leukocyte

PRR Pattern recognition receptor

RB Reticulate bodies

RPM Rotations per minute

SD Standard deviation

STD Sexually transmitted disease

STI Sexually transmitted infection

TH0 Naive T Cells

TH1 T Helper 1 Cells

TH2 T Helper 2 Cells

TNF Tumor necrosis factor

UGT Upper genital tract

WHO World Health Organization

1

CHAPTER 1. CHLAMYDIAE

Historical Perspective

Tales as old as time . . . .

-Howard Ashman and Alan Menken

Descriptions of chlamydial disease date back to ancient Chinese and Egyptian

texts detailing trachoma, a blinding chlamydial ocular disease. In 2002 Dimitrakov

reviewed the expedition in which Ludwig Halberstaedter and Stanislaus von Prowazek

discovered the causative agent of trachoma [1]. In 1907, while on the island of Java,

Halberstaedter and von Prowazek pioneered early chlamydial etiology studies by

inoculating orangutans with material obtained from trachoma patients. They were able to

demonstrate the infectious nature of the pathogen by describing intracellular vacuoles in

Giemsa-stained epithelial cells derived from conjunctival scrapings of the infected

animals but incorrectly characterized the agent as protozoan.

In 1910, Linder reported finding the same type of intracytoplasmic inclusions in

the eyes of neonates and linked their neonatal conjunctivitis to intrapartum exposure in

women with unrecognized and untreated infection. Linder went on to speculate that the

infection was transmitted sexually after identifying inclusion bodies in the mother’s

cervical scrapings, urethral cells from the fathers, and in individuals with non-

gonnococcal uretheritis [2] [3] [4].

In the 1930’s Samuel Bedson and colleagues characterized the developmental

cycle of psittacosis particles, which at the time were thought to be viruses given their

dependence on eukaryotic cells for replication. In 1957, fifty years after the Java

excursion, C. trachomatis was successfully isolated and cultured in yolk sacs by Feifan

T’ang et al [5] [6]. It was not until the mid 1960’s that chlamydiae would be defined as

prokaryotic bacteria that possessed a non-infectious intracellular replication phase [7]. In

1969, Gordon et al described culturing chlamydiae in irradiated McCoy cells, a less time

consuming technique alternative to isolating chlamydiae in yolk sac that would

revolutionize diagnostic procedures for chlamydial infection [8].

Taxonomy

What is in a name?

–William Shakespeare

Chlamydiae are prokaryotic obligate intracellular bacteria that belong to the order

Chlamydiales. Under the Chlamydiale umbrella are the families Chlamydiaceae,

Parachlamydiaceae, Waddliaceae, and Simkaniaceae [9]. In 1999, it was recommended

that the Chlamydiaceae family be subdivided into two genera, Chlamydia and

Chlamydophila, based on the phylogenetic analysis of the 16s and 23s rRNA [10] but this

recommendation was riddled with inconsistencies and controversial amongst those in the

2

chlamydial field. As reviewed by Stephens and colleagues [11], the overwhelming

majority of publications (81% in the year 2006), continued to use the single genus name

Chlamydia, despite the implementation of the two genera system. To that end, nine

species are recognized in the Chlamydia genus: Chlamydia abortus, Chlamydia caviae,

Chlamydia felis, Chlamydia muridarum, Chlamydia pecorum, Chlamydia pneumoniae,

Chlamydia psittaci, Chlamydia suis, and Chlamydia trachomatis. Historically,

chlamydiae serovars were based on conventional immunoepitiope analysis by

monoclonal antibody directed against the major outer membrane protein (MOMP) [12].

Today, chlamydial classification includes genovar sequence data of ompA, the gene that

encodes MOMP [13] [14]. The ompA-based classifying system, when compared to the

conventional sera-based analysis, more accurately reflects strain virulence and genotypic

diversity on the population level [15].

Developmental Cycle

In the circle, the circle of life.

–Sir Timothy Miles Bendon Rice

Originally described in the 1930’s by Samuel Bedson et al [16], the unique

biphasic developmental cycle of chlamydiae has been extensively reviewed [17] [18] [19]

[20] [21] [22]. Two morphologically different forms characterize the chlamydial

developmental cycle, the elementary body (EB) and the reticulate body (RB). The

elementary body is the infectious form of the organism. Although small in size, about 0.2

to 0.4 microns in diameter, EBs are resistant to extracellular conditions and are able to

attach and enter susceptible host cells. Once endocytosed into the host cell, EBs

differentiate into RBs inside a membrane bound vacuole called an inclusion. Reticulate

bodies are the metabolically active and non-infectious intracellular forms of chlamydiae

which multiply via binary fusion within the inclusion. After repeated cycles of cell

division, RBs undergo a second differentiation stage resulting in infectious chlamydial

EBs. Depending on the Chlamydia species, infectious progeny exit the initially infected

cell between 30-72 hours post infection and infect neighboring host cells (Figure 1-1).

For millions of years Chlamydia species have infected eukaryotic cells and many

researchers believe that they have ensured their survival by deviating from their normal

biphasic developmental cycle. Persistence, a reversible interruption in the productive

intracellular chlamydial growth cycle by environmental factors, has been reviewed many

times over [23] [24] [25] [26] [27] and is characterized by large aberrant non-replicating

RBs which are unable to alternate between EB and RB morphological forms. In vitro

inducers of this abnormal growth state include physiological changes in the host cell,

gamma interferon treatment [28] [29] [30] [31] [32], beta lactam administration [33] [34]

[35], nutrient restriction [36] [37] [38] [39] [40], and concurrent herpes infection [41]

[42] [43]. By definition, restoration of normal chlamydiae development occurs when the

environmental stressor that induced the persistent state is removed. While the likelihood

of persistence being involved in chlamydiae-induced pathogenesis has been documented

in a variety of culture-based systems, particularly as it relates to antibiotic treatment and

3

Figure 1-1. Chlamydiae developmental cycle.

Note: Chlamydiae share a unique biphasic developmental cycle consisting of the

following steps: 1) Attachment and entry of the infectious Elementary Body (EB) form 2)

Primary differentiation from the infectious EB form to the metabolically active but non-

infectious Reticulate Body (RB) form in a cytoplasmic vacuole called an inclusion 3)

Cell division by binary fission 4) RB genomic replication 5) Secondary differentiation

from RB to EB and 6) Cell lysis/endocytosis and release of infectious progeny.

Chlamydiae may enter an abnormal state of growth called persistence under the selective

pressure of a variety of inducers. Chlamydiae re-enter the cycle and resume normal

development when the selective inducer is removed.

1. Attachment and

Entry

2. Primary

Differentiation

3. Cell

Division

4. Genomic Replication

5. Secondary

Differentiation

6. Release

Persistence

4

host immunological pressures, conclusive evidence of human chlamydial persistence

remains to be demonstrated.

Chlamydiae Clinical Significance

Chlamydial infection is of global importance given its broad host tropism, the

scope of its geographic distribution, and the risk of developing debilitating sequelae. Of

the nine Chlamydia species, three are known to cause a diverse spectrum of diseases in

human populations: Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia

trachomatis (Table 1-1).

Chlamydia pneumoniae

So you’re telling me that you can catch Chlamydia by just breathing? There goes the

neighborhood.

–Corey Dannard

Chlamydia pneumoniae was introduced as a novel member of the genus

Chlamydia by Grayston et al in 1989 and is commonly associated with upper respiratory

infections. C. pneumoniae causes approximately ten percent of community-acquired

pneumonia and five percent of pharyngitis, bronchitis, and sinusitis [44]. In addition,

strong associations exist between C. pneumoniae infection and atherosclerosis, a chronic

cardiovascular disease whose complications lead to half of the adult deaths in the United

States and other parts of the western world (Reviewed by Belland et al [45]). It is

important to note that the association between Chlamydia pneumoniae and

atherosclerosis is a point of debate given the failure to improve clinical outcomes with the

administration of antichlamydial antibiotics in large scale clinical trials including patients

with cardiovascular disease [46] [47] [48].

Chlamydia psittaci

You do know, of course, that zoonotic doesn’t mean the viruses came from the zoo.

–Law and Order: Criminal Intent

Chlamydia psittaci, a zoonotic pathogen whose natural reservoir is avian, is

recognized by the Centers for Disease Control and Prevention as a category B select

agent due to the ease of respiratory dissemination and associated morbidity and mortality

rates [49] [50]. As reviewed by Harkinezhad and colleagues, human C. psittaci infection

is called psittacosis and is acquired by inhalation or, to a lesser extent, ingestion of bird

excretions [51]. After inhalation, the organism infects the respiratory epithelium and

remains latent for up to three weeks void of clinical symptoms. Following the incubation

period those infected experience flu-like symptoms such as headaches, chills, fever,

cough, and in rare instances, neurological and cardiac-related complications. In extreme,

untreated cases the infection can be fatal.

5

Table 1-1. Chlamydiae that cause human disease.

Species Serovar Acute Disease Sequelae

C. pneumoniae Community-

Acquired

Pneumonia

Arthritis

Sinusitis Asthma

Bronchitis Arthrosclerosis*

Pharyngitis

C. psittaci Atypical Pneumonia Potentially Fatal

Renal and Hepatic

Complication

C. trachomatis A, B, C Conjunctivitis Trachoma

D,E,F,G,H,I,J,K Cervicitis, Urethritis Pelvic Inflammatory

Disease (PID)

Ectopic Pregnancy

Involuntary

Infertility

Reiter’s Syndrome

LGV Lymphogranuloma

venereum (Bubonic)

Fitz-Hughes-Curtis

Syndrome

Note: * indicates debated chlamydial-sequelae association.

6

Chlamydia trachomatis

Chlamydia trachomatis is composed of four biovars, biological strains, based on

the target cells they infect and whole genome sequencing [52] [53] [54]. As implied by

the names, the ocular trachoma lineage commonly infects eye mucosa while urogenital

linages most commonly infect the genital epithelia. Lymphogranuloma venereum (LGV)

is a disseminating biovar that thrives within the lymphatic niche. Trachoma biovars are

subdivided into fifteen serovars based on ompA antigenic variation encoding the major

outer membrane protein (MOMP). Loosely, serovars A, B, Ba, and C serve as the

causative agent of Trachoma, the leading cause of infectious blindness throughout the

world. Serovars D-K are commonly associated with sexually transmitted infection. While

L1, L2, L3 are the three main LGV serovars, the newly identified L2b serotype has

emerged as the causative agent of the current European and North America epidemic

[54].

Ocular Chlamydia trachomatis

The earliest documented historical accounts of chlamydial infection detail

trachoma, a contagious disease of the conjunctiva, the outside covering of the eye, and

cornea caused by Chlamydia trachomatis. While trachoma rarely occurs in western

societies, ocular serovars of C. trachomatis are epidemic in parts of Africa, Asia, South

America, Australia, and the Middle East making it the world’s leading cause of

preventable infectious blindness.

Active infection, a self-limiting inflammation of the conjunctiva, is primarily seen

in children and is transmitted by direct contact with mucosal secretions, poor sanitation,

inanimate objects such as towels and clothing that have infected secretions on them, and

natural vectors such as flies (Reviewed by [55] [56]). Recurrent infection, which is

common in developing countries, leads to the development of scarred tissue and,

eventually, trichiasis, inversion of the eyelid. Inwardly turned eyelashes cause physical

damage to the cornea. This results in the blindness seen primarily in endemic adult

populations. The World Health Organization endeavors to eliminate trachoma by the year

2020 with the implementation of the S.A.F.E. campaign which includes surgery,

antibiotics, facial cleanliness, and environmental improvements [57] [58].

Genital Chlamydia trachomatis

Sexually transmitted diseases are hidden epidemics of tremendous health and

economic consequence in the United States…the scope, impact and consequences of

STDs are under recognized by the public and health care professionals.

–Institute of Medicine, 1997

Sexually transmitted Chlamydia trachomatis has a significant impact on human

health given its adverse effects on reproduction. The World Health Organization

7

estimates that ninety million cases of chlamydial infection occur worldwide each year

and an estimated four million cases are reported annually in the United States. Many

believe these estimates are much lower than the actual incidence due to the fact that

infection is largely asymptomatic. While individuals who are infected may not experience

symptoms, it is important to note that they are still at risk of developing long term

sequelae.

In men, C. trachomatis primarily infects the urethra making it the most common

cause of non-gonococcal urethritis. In some instances, the infection spreads from the

urethra to the epididymis resulting in epididymitis, a condition primarily associated with

sexually active males under the age of thirty-five. Infection may also result in Reiter’s

Syndrome in male and female populations. Whether or not Chlamydia infection plays a

direct role in male infertility is still controversial despite the fact that chlamydial DNA

can be recovered from a substantial number of male partners in infertile couples [59] [60]

and has been recovered attached to spermatozoa from the peritoneal fluid of women with

salpingitis [61].

Genital serovars of chlamydiae are of particular importance to women due to the

irreversible reproductive sequelae that may result post infection. Infection of the cervix

can ascend causing endometritis, inflammation of the endometrium, and salpingitis,

inflammation of the fallopian tubes. Untreated C. trachomatis infection has been linked

to chronic complications such as Pelvic Inflammatory Disease (PID), involuntary

infertility and ectopic pregnancy. Moreover, chlamydial infection increases the risk of

contracting human immunodeficiency virus (HIV) and has been implicated in the

development of human papilloma virus (HPV)-induced cervical neoplasia [62] [63].

Although rare, C. trachomatis-induced salpingitis spreading beyond the upper genital

tract into the peritoneum has been documented. The resulting peritonitis and perihepatitis

is called Fitz-Hugh-Curtis Syndrome and may be accompanied by upper quadrant

abdominal pain and the development of adhesions that resemble the strings of a violin

[64] [65].

Mouse Model of Genital Infection

Life is hard for insects. And don’t think mice are having any fun either.

–Woody Allen

Humans are not the only hosts in which chlamydiae can establish an infection and

cause disease. Guinea pigs, turkeys, sheep, and higher order primates have been used to

study chlamydiae-associated disease but an extensive amount of data has been extracted

from mouse models. Mice infected with either C. muridarum or human biovars of C.

trachomatis are most frequently used as models of chlamydial genital infection due to the

ease of reproducibility afforded by inbred strains, commercial ability of reagents, and

genetically engineered animals that allow for immunological interrogation [66].

While nonhuman primate [67] [68] [69] and guinea pig [70] [71] models of

infection were established, Barron and colleagues [72] developed a novel system in

8

which researchers intravaginally infected mice with Chlamydia muridarum, previously

known as C. trachomatis mouse pneumonitis strain or MoPn, in 1981. In the Barron

study, chlamydial inclusions were identified by examining Giemsa stained vaginal smear

preparations and chlamydiae-specific immunofluorescent cervical scrapings and

epithelial tissue. These findings were significant because they identified a convenient

model using a natural mouse pathogen that induced pathologies remarkable similar to

those observed in humans infected with C. trachomatis serovar D [73].

Mouse models of chlamydial infection have been used to evaluate the role a

host’s genetic background may play in chlamydiae infection resistance and Chlamydia-

associated outcomes. De la Maza and colleagues intravaginally infected mice with

varying H-2 complexes to determine its effect on chlamydial-related infertility [74]. The

H-2 complex defines the major histocompatibility complex (MHC) in mice and is

homologous to HLA in humans. Six weeks after challenge BALB/c, C57BL/6, and C3H

mice were mated with male breeders and the embryos were counted. Seventy-five

percent (N=20) of C57BL/6 mice became pregnant and had a mean of 4.5 embryos per

mouse. Forty percent (N=20) of BALB/c mice became pregnant and had a mean score of

1.5 embryos per mouse. Thirty percent (N=20) of C3H animals became pregnant and had

a mean score of 1.7 embryos per mouse. From these studies the researchers concluded

that the genetic makeup of the host modulates the degree of chlamydial-induced

infertility. In 1997, Darville et al expanded the study by comparing C3H/HeN mice with

C57BL/6 mice using varying strains of Chlamydia [75]. When intravaginally infected

with C. trachomatis, serovar E or C. muridarum, C3H mice had an increased incidence of

hydrosalpinx, increased chlamydial shedding, and prolonged infection course when

compared to C57BL/6 animals. This suggested that genetic factors played a role in

chlamydial resistance and that the murine model could be used to understand the

mechanisms responsible for resistance variability in the human population.

Advances in human genetics, such as the human genome project, have

revolutionized our understanding of the host’s role in human health and disease by

allowing for inter- and intra-species genetic comparisons [76] [77]. Indeed, whole

genome association studies would allow researchers to interrogate genes and their

associated disease phenotypes relatively quickly when compared to the previous method

of “knocking out” genes in in vivo models and looking for changes in the initially

observed response. As one would expect, identifying conserved genetic sequences across

diverse species would require vast amounts of genetic information. This requirement

grants the mouse model a significant advantage over other animal models given the fact

that genetic sequences and many gene function relationships have been identified and are

readily available using informatics tools like the Mouse Genome Database

(http://www.informatics.jax.org/).

Although chlamydial comparative studies are still in their infancy, high-

throughput genomic analyses have the potential to transform how we currently identify

and therapeutically treat those infected. As proof of principle, Miyairi and colleagues

recently sought to predict outcomes of systemic chlamydial infection using recombinant

inbred mice (BXD) and computational modeling [78]. Infection of parental strains,

9

C57BL/6 and DBA/2J, an extensive panel of B (C57BL/6) x D (DBA/2J) mice, in

conjunction with gene mapping and computational Bayesian network modeling were

used to define underlying pathways contributing to variations in disease severity. The

researchers validated predictions that Ctrq3 or polymorphisms in immunological relevant

GTPases conferred resistance in B6 dominant genetic backgrounds, whereas,

susceptibility was heightened in D2 dominant backgrounds as a function of neutrophilic

influx modulation. While there are no homologs of interferon-inducible p47 GTPases in

humans, Miyairi’s findings implicate neutrophils as a tentative therapeutic target, validate

computational chlamydiae-related modeling as a way of predicting disease outcomes, and

highlight recombinant inbred strains as a way of elucidating previously unknown host-

derived pathways contributing to disease.

Murine Genital Tract Pathology

C. muridarum, although originally isolated from the murine respiratory tract [79]

[80], closely mimicked human sexually transmitted chlamydiae disease when used to

infect the genital tracts of mice. In 1983, Swenson and colleagues reported Chlamydia-

induced genital pathology mirrored that seen in human populations [81]. In these

experiments mice were inoculated with C. muridarum in the ovarian bursa, a thin

membrane that encapsulates the ovary and separates it from the interperitoneal cavity.

Hydrosalpinx, blockage of the fallopian tube(s) with serous fluid, was observed in mice

between 25-30 days post infection. Salpingitis and hydrosalpinx formation have been

linked to involuntary infertility in human female populations by irreversible scarring in

the reproductive system and similar outcomes were demonstrated in the model. Although

the natural route of infection was not used in this study, it validated that the mouse model

could be used to explore mechanisms associated with the development and severity of

chlamydiae-induced upper tract complications. Since, several studies, including those

outlined in this body of work, have demonstrated vaginal inoculation of the mouse results

in reproducible upper genital tract pathology.

Murine Model Limitations

Although the murine model has been advantageous, there are some caveats. For

instance, there is great variability in chlamydiae strains, innoculum doses used, and the

inbred strain used. The efficiency of infection using human strains of C. trachomatis is

significantly lessened when compared to the strain isolated from the murine respiratory

tract, C. muridarum [82]. As a result, investigators commonly use higher doses of

Chlamydia trachomatis to establish murine infections. Inoculating doses have also been a

point of debate in the chlamydial field. In 2004, Maxion and colleagues evaluated

differences in BALB/c cell infiltration and pathology formation as a result of innoculum

doses ranging from 104 to 107 inclusion-forming units (IFUs). They found that dose

variation altered immune cell representation in the genital tract noting increases in PMN

and DC infiltrates in the lower genital tract as chlamydial dose increased [83]. Carey et al

investigated the effects of inoculum dose on pathology development in BALB/c female

10

mice [84]. They concluded higher doses of Chlamydia muridarum lead to greater oviduct

infection.

Lastly, and perhaps the most relevant argument, is that C. trachomatis is

transmitted by oral, vaginal, and anal sexual contact with an infected individual. The

likelihood of large amounts of infectious organisms being transmitted by the routes

mentioned is low. In support of this argument, the infectious chlamydial load in humans

is low with a median IFU of 72 from male-derived urethral swabs and 450 IFU from

cervical swabs taken from women [85]. Collectively, these studies underscore the need to

standardize infection parameters in chlamydial models across the board to reflect the

likely transmission inoculums seen in human scenarios of infection.

Another caveat of the mouse model is the five day estrous cycle. The frequency of

epithelial uterine sloughing proved to be a problem in establishing chlamydial infection

in mice because the target population (epithelial cells) was turning over prior to the

completion of the chlamydial developmental cycle. Tuffery et al performed experiments

that showed the female genital tract epithelium could be stabilized by subcutaneously

injecting progesterone one to two weeks prior to intravaginal infection (Tuffrey, and

Taylor-Robinson, 1981). Hormone treatment stabilized murine menses and enhanced

chlamydiae genital infection by preventing the normal renewal of genital epithelium. As

a result, the mouse model can be used to study the natural course of infection and

chlamydiae-induced pathology with increased reproducibility. Currently, the use of

progesterone in animal models is debated because sex hormones have been shown to

affect susceptibility to a number of sexually transmitted infections in human and animal

studies [86] [87] [88].

Another concern is the availability of a persistent murine model. In human

female populations, persistent infections defined by chronic asymptomatic chlamydial

genital tract infections, may give way to the development of reproductive complications

such as PID, chronic abdominal pain, and tubal infertility. As mentioned previously, in

vitro persistent states have been induced by environmental stresses such as the interferon

gamma inducible tryptophan decyclizing enzyme 2, 3-indoleamine dioxygenase, iron

depletion, and by treatment with penicillin. In efforts to reproduce persistence in vivo,

Ramsey et al used iNOS knockout mice [89]. When NOS2-/- mice were infected with C.

muridarum, they exhibited higher rates of upper genital tract sequelae but culture-based

resolution was comparable to that observed in wild-type mice. In 1997, Cotter et al

intravaginally infected wild-type immunocompetent mice and were able to reactivate

chlamydiae shedding after initial clearance, evidenced by the inability to recover viable

organisms from the vaginal vault, with the immunosuppressive drug cyclophosphamide

[90]. While these studies illustrate persistence in the mouse, the mouse model may not

be a useful tool for exploring mechanisms of persistent infection because it difficult to get

reactivation. Further investigation for a suitable animal model is warranted given the lack

of conclusive evidence for persistent infection in the human population.

11

Immunological Response to Genital Infection

Before discussing the host immune response to sexually transmitted chlamydial

infection, it is important to note key distinctions of the female genital mucosa when

compared to other mucosal sites of the body. The reproductive environment is unique in

that it must exhibit a certain level of tolerance for commensal flora that colonize the

lower genital tract and withstand the presence of an immunologically foreign fetus in the

uterus during pregnancy, all the while maintaining its ability to mount a response to

pathogenic organisms. In addition, the genital environment differs from other mucosal

sites like the proximal intestinal system in that many of its effector functions, including

immunological properties, are hormonally regulated [91]. These distinctions and the lack

of local concentrations of lymphoid tissue such as the gut-associated lymphoid tissue

(GALT) component Peyer’s Patches in the intestine or bronchular-associated lymphoid

tissue (BALT) in the lung contribute to the complexity of chlamydiae infection in the

genital tract.

Intracellular Immune Response Overview

After “self verses non-self” discrimination, the host orchestrates appropriate

immunological responses based on the niche in which pathogens thrive [92]. Bacteria

such as Mycobacterium tuberculosis[93] [94], species associated with the genus

Rickettsia [95] [96], chlamydial species [82] [97] [98] [99] [100] [101], and viruses like

HIV [102] [103] and influenza [104] are hallmark intracellular pathogens that require

specialized approaches to achieve clearance. Antigen-presenting cells (APCs) and T-cells

are crucial to intracellular pathogen elimination. For instance, pattern recognition

receptors (PRRs) associated with APCs like dendritic cells or macrophages recognize

pathogen-associated molecular patterns (PAMPs) which, in turn, initiate antimicrobial

compounds such as interferon-gamma, tumor necrosis factor-alpha, and interleukin-two

[105]. These cytokines assist in the activation of other APCs and push naïve T-cells

(Th0) toward an appropriate TH1 pathogen-specific linage. This cascade culminates in B-

cell activation and the development of plasma cells, antibody-producing B-cells that

ready the host for subsequent encounters.

Chlamydia Innate Response

Induction of the innate immune response is central to mounting an effective attack

against chlmaydial genital pathogens. The process begins with the recognition of

pathogen associated molecular patterns (PAMPS) by Pattern Recognition Receptors

(PRRs) of Antigen Presenting Cells (APCs) or host cell membranes. Various chlamydiae

components or the entire organism may serve as ligands for toll-like receptors (TLRs), a

membrane bound family of PRRs. For example, chlamydiae-derived lipopeptide was

shown to stimulate TLRs 2, 1, and 6 in macrophages [106]. Although less stimulatory

than E. coli lipopolysaccride (LPS), chlamydial LPS may serve as a ligand for TLR4

[107] and to a lesser extent TLR2 [108]. Moreover, whole organism was used to

determine differences in chlamydiae-induced oviduct pathology after discriminatory

12

stimulation of TLRs 2 and 4 [109]. Stimulation of TLRs give way to activation of the

NF-K B pathway which, in turn, results in production of pro-inflammatory cytokines and

chemokines.

Almost immediately after infection, a cascade of proinflammatory cytokines are

secreted by the target epithelium. This was first reported by Rasmussen and colleagues in

1997 [110]. Using the in vitro HeLa 229 epithelial cell line, these studies showed that

interleukin-8, growth-related oncogene- alpha (GRO-alpha), neutrophil-derived

granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-6, and

interleukin-1alpha were secreted following infection. Experiments using mouse derived

oviduct epithelial cells performed by Johnson et al showed that proinflammatory

cytokines and chemokines like tumor necrosis factor alpha, GM-CSF, interleukin-6, MIP-

2, KC, MCP-1 and MCP-5 were produced following C. muridarum infection [111]. The

secretion of these effector molecules lead to the massive influx of innate immune cells.

Darville and colleagues, by flow cytometry, and Morrison and Morrison, by in situ

immmunohistochemistry, characterized the primary infiltrate and found that monocytes,

natural killer cells, and neutrophils were prevalent in these cell populations [75] [112].

The newly recruited cells expanded the repertoire of effector molecules being produced

and, depending on the cytokines and chemokines released, recruited pathogen-specific

lymphocytes to the site of infection.

Chlamydia Adaptive Response

Effective host responses require the induction of, both, the innate and acquired

arms of the immune system. Indeed, resident and recruited innate immune cells such as

neutrophils and professional antigen presenting cells such as macrophages and dendritic

cells begin producing tumor necrosis factor-alpha, interleukin-12, and interferon-gamma.

This stimulates naïve T cells toward CD4 T-helper 1 lineages which are necessary for

chlamydial clearance in the genital tract. Studies in interferon gamma [113] and

interferon gamma receptor knockout mice [114] illustrate a prominent role for the

cytokine in the resolution of primary chlamydial infection. Rank and colleagues first

demonstrated an important role for CD4 T cells in the genital tract by vaginally infecting

athymic nude mice [115]. Nude animals had an active infection evidenced by chlamydial

shedding for more than 200 days post infection, whereas animals with intact T cell

populations resolved infection within 21 days. Landers et al reinforced this finding in

1991 when they used CD4 antigen specific anti-L2T4 antibodies to deplete CD4 in mice

vaginally infected with C. muridarum [116]. These depletion studies resulted in

increased vaginally shedding of Chlamydia and an increased number of organisms

recovered from the oviduct. Su et al later transferred CD4 and CD8-enriched spleen cells

from immune mice to naïve mice and found that adoptive transfer of CD4 lymphocytes

conferred immunity in C. muridarum genital tract infection [117]. Kelly and colleagues

published work illustrating that CD4+ cells were abundantly recruited to the genital tract

following intravaginal infection due to interaction between CD4+ cell home receptor

alpha-4-beta-7 and adhension molecules, ICAM-1, VCAM-1, and MadCAM-1 [118]

[119]. Most recently, Gondek and colleagues reported that CD4+ T cells were necessary

13

and sufficient to clear genital tract infection [120]. Using transcervical inoculation, a

method that directly infects the uterine lining of mice, the researchers demonstrated C.

trachomatis, LGV and C. muridarum infected mice are protected from infection and

reinfection when treated with pathogen-specific CD4+ lymphocytes. Furthermore, when

animals were treated with anti-CD4 antibody, Chlamydia 16S DNA levels where

comparable to those observed in naïve mice. Collectively, these studies demonstrate the

importance of T helper 1 type cytokines and CD4 T cells in the resolution and protection

of chlamydial-induced genital infection.

The humoral response has also been implicated in chlamydial immunity. Plasma

cells, commonly known as antibody producing B cells, are thought to play a leading role

in protecting against reinfection, while playing a secondary role to CD4-mediated

resolution of primary infection [99]. One mechanism by which B cells may control

subsequent infection is by antibody driven neutralization, which is plausible given the

biphasic developmental cycle of chlamydiae. Data from Peeling et al using serum from

guinea pigs to neutralize C. trachomatis with UM-4, a strain specific monoclonal

antibody in an in vitro neutralization assay support this theory [121]. Furthermore,

Morrison and colleagues showed that B-cell deficient mice, when depleted of CD4+ T

cells during secondary infection, where unable to clear infection [98] [99]. Although the

experiments by Morrison et al did not rule out the feasibility of direct neutralization, they

implicated a more collaborative mechanism between CD4 lymphocytes and plasma cells,

perhaps by the enhancement of antigen presentation during recurrent infection episodes.

Although the exact mechanism remains unclear, antibody, particularly IgG given it is the

predominant antibody produced in the genital mucosa, is thought to play a collaborative

role in chlamydial immunity.

Vaccination

The prevention of disease today is one of the most important factors in the line of human

endeavor.

–Charles H. Mayo, M.D.

The immune response is the host’s natural defense mechanism to foreign antigens

but the system can be manipulated to respond to foreign antigens quicker and more

efficiently than if it were the first encounter. The previously mentioned form of

manipulation serves as the basis for vaccination or, what some refer to as immunization.

Although current anti-chlamydial therapies effectively clear the organism from

the lower genital tract, they do not address the potential consequences of reinfection.

Batteiger and colleagues reported that almost one third of previously infected individuals

are reinfected with the same serovar due to sexual interaction with the same sexual

partner [122], potentially increasing the likelihood of irreversible tissue damage. One

way of addressing reoccurring infection is through the development of an effective and,

most importantly, safe vaccine.

14

Perhaps the most well-known C. trachomatis vaccination studies were the

trachoma human trials performed in the 1960’s [123] [124]. During these trials children

were intramuscularly vaccinated with formalin-fixed whole organisms and traced for

three years. Unfortunately, a portion of those immunized developed severe disease upon

exposure to chlamydiae while others developed partial, serovar-specific immunity when

compared to unvaccinated controls. Since then, vaccination studies have been exclusively

performed in animal models and frequently exploit components of chlamydiae for

vaccination.

First purified by Caldwell and colleagues [125], the major outer membrane

protein (MOMP) has been the focal point of chlamydiae subunit vaccination studies for

the past thirty years. Recently reviewed by Farris and Morrison, native or recombinant

protein, DNA, plasmid, and outer membrane complexed MOMP have been the most

frequently studied MOMP-derived antigens [126]. At best, these studies have only

elicited partial immunity by either failing to reduce bacterial burden, failing to protect

animals from reinfection, and/or failing to protect against sequelae formation.

Interestingly, recombinant vault nanoparticle delivery of MOMP immunogens has

renewed hope in MOMP’s potential as an effective vaccine candidate. Intranasal

immunization with rMOMP nanoparticles results in a T-helper 1 driven immune response

and significantly reduces chlamydial vaginal shedding when compared to animals

immunized with live C. muridarum [127].

Additional antigens have been identified based, mainly, on human seroactivity.

Some of these include chlamydial protease-like activity factor (CPAF), other outer

membrane proteins such as OmcB and Pmps, porin protein B (PorB), and the type III

secretion protein, Tarp. Of the previously mentioned chlamydial components, CPAF was

thought to have the most promise. Intranasal immunization with recombinant CPAF

(rCPAF) plus interleukin-12 reduced bacterial shedding and sequelae formation in

BALB/c intravaginally challenged with C. muridarum [128]. In addition, rCPAF and

Cpg-1826 was used to intranasally immunize BALB/c mice prior to multiple rounds of C.

muridarum intravaginal challenge [129]. rCPAF plus CpG vaccination resulted in a

significantly greater number of pregnancies when compared to mock immunized controls

indicating protection post primary and secondary exposures to genital chlamydial

infection. In 2012, Chen et al called into question the entire body of work done using

CPAF and its proposed substrates after reporting CPAF lost its ability to cleave or

degrade 11 of the 16 previously reported host proteins when a CPAF- specific inhibitor

was used prior to cell lysis [130]. These results suggest that the proteolysis activity

observed in all previous CPAF studies was likely due to the way in which the

preparations were prepared and discredit, at least for now, the idea that CPAF protease

activity is a virulence factor important in chlamydial pathogenesis.

Control Measures

Chlamydia trachomatis infection is the most commonly reported sexually

transmitted disease in the United States and a significant threat to public health world-

wide. Chlamydia control programs were implemented to alleviate the public health

15

burden of chlamydial infection by improving detection mechanisms, shortening the

duration of infection, and tracking source-associated sexual networks [131]. These

programs ushered in an initial lag phase denoted by a decline in chlamydial cases.

However, over the last two decades, case notifications of Chlamydia-associated infection

have steadily increased and, in many countries, exceed rates recorded prior to the

implementation of intervention strategies [132] [133] [134]. Norway, Finland, Sweden,

and Canada have documented similar trends with an initial decline following the

introduction of Chlamydia control programs [132] [135] [136] [137]. In recent years,

these countries have witnessed chlamydial rates exceeding those recorded prior to the

establishment of surveillance systems. Given the magnitude and scope of current

epidemiological trends, researchers and policy makers alike are interested in pinpointing

the causal agent(s) for increased chlamydial case notifications.

Sexually Transmitted Chlamydia Epidemiology

The insidious nature of Chlamydia trachomatis infection has made it the world’s

most common cause of curable sexually transmitted disease. In 2010, over a million cases

(1,307,893) of chlamydial genital tract infection were reported to the Centers for Disease

Control and Prevention from fifty states and the District of Columbia [63]. This was the

largest number of cases reported to the CDC for any reportable condition with a case rate

of 426.0 per 100,000 people, representing a 5.1% increase in reported cases over the

previous year. Although these rates are staggering, the actual number is more than likely

higher than that reported seeing as many cases remain undiagnosed.

Gender disparities have consistently been reported among individuals infected

with sexually transmitted C. trachomatis. According to the National Sexually Transmitted

Disease Surveillance Report 2010, women bear a heavier Chlamydia burden than men

with reported rates two and half times more than their male counterparts despite the fact

that Quinn and colleagues reported a nearly identical frequency of transmission among

494 male and female sexually transmitted disease clinic patients [138]. This discrepancy

may be attributed to the fact that women are more likely than men to be screened for

chlamydial infection [139]. However, the switch from urethral swabbing to urine-based

non-invasive nucleic acid amplification testing as the main detection method may result

in more men opting to get tested [140] [141]. This is supported, at least in part, by the

36% increase in chlamydiae case notification since the year 2006. A 19.5% increase was

observed in the female population between 2006 and 2010, indicating the increase in

male reporting was not solely because of an increase in population-based chlamydiae

prevalence [63].

Age has been implicated as a risk factor for urogenital chlamydial infection [142]

[143] [144] [145] [146] [147] [148]. The highest notification rates correspond to sexually

active female adolescents between the ages of 15-19. The second highest rate among

females is young adults between the ages of 20-24. Age-specific rates among males are

higher among 20-24 year olds with 1,187 cases per 100,000 people reported in the year

2010. These numbers are particularly troubling considering chronic chlamydial

16

complications have been linked to infertility in both genders and the age groups greatest

affected represent peak reproductive years [63].

Significant ethnic and racial disparities exist among reported sexually transmitted

infections, specifically Chlamydia cases. For instance, African-Americans only represent

14% of the national demographic, but they account for approximately half of all reported

syphilis and chlamydiae cases and 75% of all reported gonorrhea infections [63]. With an

incidence rate 1,167.5 per 100,000 persons, African-American chlamydial rates are eight

times higher than white Americans. American Indian/Alaska Natives chlamydial rates

are 4.3 times higher than white Americans while Hispanic American chlamydial rates are

2.7 times greater than those reported among Caucasian-Americans. While it would be

easy for researchers and epidemiologists to dismiss these health inequalities based on the

idea that these communities are more susceptible to infection and/or are more likely to

participate in risky behaviors, the reasons are more complex [149]. While individual

behaviors can influence health, surveillance trends are, in large part, influenced by

cultural, economical, environmental, educational, and social factors [150] [151] [152].

For instance, in a study conducted by Kaplan and colleagues in 77 different Chicago

communities, sexually transmitted infections were higher in neighborhoods that had high

poverty rates, high unemployment rates, and a low percentage of high school graduates

[153]. In a cross-sectional analysis of over 12,000 young adults, Nguyen et al concluded

that geographical location and economic status were factors associated with chlamydial

reporting with study participants living in the southern region and no functional income

being more likely to report chlamydiae infection [154]. While the studies citied highlight

findings seen in relatively small participant groups, they are representative of health

determinants influencing national surveillance profiles.

Hypotheses for Rebounding Chlamydia Rates

Seven hypotheses were put forth to explain this phenomena and were published

by Rekart and Brunham [133] and are adapted in Table 1-2. Hypotheses one through

four relate to the use of nucleic acid amplification testing for detecting genital infection

with chlamydiae. Hypothesis one speaks to an increased probability of “false positive”

tests because of a loss of specificity in detection methods. Watson and colleagues

performed a meta-analysis including the following diagnostic methods for urogenital

chlamydia: nucleic acid amplification testing (NAAT), gene probe, enzyme immunoassay

(EIA), direct immunofluorescence (DFA), and cell culture [141]. The study concluded

that NAAT testing was the best option for massive screening programs because the

results were comparable to those observed using the “gold standard”, cell culture, and

urine samples did not constitute a gender bias. Men and women would be willing to

undergo screening using this non-invasive technique.

Hypotheses two through four argue increased sensitivity, improved screening

amongst high-risk populations, and greater screening among the male population are the

basis for surveillance data trends. In 2006, Burckhardt et al assessed the impact of

changing from culture-based chlamydial detection to nucleic acid amplification testing in

17

Table 1-2. Increased chlamydial rate hypotheses.

Hypothesis

Number

Hypothesis Description

H1 False positive increase due to lower specificity of NAAT methods

compared to culture methods

H2 Increase in case detection due to increased sensitivity of NAAT testing

compared to non-NAAT testing

H3 Higher testing rates among men due to non-invasive NAT urine-based

testing

H4 Targeted screening of high-risk populations and NAAT self-collected

sampling among female population(s)

H5 Development of antibiotic resistance

H6 Increase in unsafe sexual behaviors

H7 Chlamydial arrested immunity

18

a genitourinary medicine clinic cohort [155]. Although an initial increase in positive

testing occurred in the two and half years immediately following the implementation of

NAAT testing, positive test percentages returned to those observed prior to the detection

method change. The researchers concluded that the upturn of chlamydial incidence was

not exclusively due to the improvement of detection methods.

Hypothesis five suggests antimicrobial resistance is responsible for the

rebounding rates. Indeed, antibiotic resistance is a serious issue, particularly for

individuals infected with syphilis and/or gonorrhea [156] [157] [133] [158], two major

sexually transmitted infections. Although tetracycline resistance has been documented in

chlamydiae serovars that infect livestock [159], resistance among human isolates are

rarely observed.

Hypothesis six implicates the safe sex practices, or lack thereof, as a potential

cause. Undoubtedly, the behavioral choices made by individuals influence population-

based surveillance data. Perhaps the ideal example is that of HIV/AIDS. After the initial

recognition of HIV in the early 1980’s, huge awareness campaigns were implemented

that led to a decline in STI transmission across the board [160] [161]. After effective

treatment was introduced and the general population no longer viewed HIV/AIDS as a

“death sentence”, STI incidence rates rebounded. This demonstrates that effect of human

behavior on modulating infection cases and confirms the likelihood of current trends

being a result of several factors.

The final hypothesis is the arrested immunity hypothesis. Although it is important

to remember that none of the previously mentioned postulates are likely to be mutually

exclusive, the arrested immunity hypothesis is the cornerstone of my dissertation research

and the primary theme hereafter.

Arrested Immunity Hypothesis

The arrested immunity hypothesis asserts that prompt treatment of genital tract

infection is coupled with a reduction in naturally acquired immunity, resulting in

increased incidence and prevalence of chlamydial infection [132]. Interference with the

development of host immunity to chlamydiae genital infection has been shown using the

urogenital murine model by the Caldwell research group [162]. Su and colleagues treated

C57BL/10 female mice with doxycycline at different timepoints postinfection. No IgG

antibody was detected in sera from animals that received treatment at day zero, the onset

of infection. This demonstrated the effects of early treatment on the development of anti-

chlamydial immune responses.

Brunham et al applied the Cox proportional hazards model to evaluate the effects

of antibiotic treatment on the development of population-based immunity [163]. The Cox

model is a statistical survival model developed by Sir David R. Cox that relates the time

between an “event” occurring to one or more covariates that make also occur within that

time frame. The Brunham model is hinged on findings by Molano and colleagues [164]

suggesting immunity in human populations may take years to develop. These findings,

19

when considered with the early treatment component of control programs, suggest that

human hosts don’t develop an adequate immunological response to chlamydiae due to the

truncated exposure time with chlamydial antigen(s) in the genital tract. Using the Cox

model, Brunham’s groups concluded that early treatment renders the general population

more susceptible to reinfection.

Dissertation Rationale

Despite the implementation of wide-spread control initiatives, sexually

transmitted Chlamydia rates continue to rise and are coupled with a decline in

chlamydiae-associated chronic complications such as PID, ectopic pregnancy, and

involuntary infertility [131]. Several rebounding hypotheses have been put forth to

explain current surveillance trends and, optimally, humans studies would provide the

most insight as to why intensive screening and treatment strategies are, seemingly,

ineffective. For obvious reasons, these studies are impractical given the ethical concerns

surrounding trials undertaken with the potential of chlamydial-induced upper genital tract

complications. Taking into account current therapeutic intervention, immune arrest and

infection history, we used the chlamydiae urogenital mouse model to determine the

effects of antibiotic treatment on duration of infection, immunological parameters and,

most importantly, chlamydial-induced upper tract complications in primary and

reoccurring cycles of infection. Moreover, we documented the effects of host factors,

such as age and genetic make-up, and immunization on the development of severe

chlamydial-induced genital tract disease.

20

CHAPTER 2. ARRESTED IMMUNITY: IMPACT OF EARLY TREATMENT

ON UPPER GENITAL TRACT SEQUELAE

Introduction

Sexually transmitted Chlamydia trachomatis is the most commonly reported

notifiable infection in the United States [147]. Although up to 75% of women and 50% of

men with lower tract chlamydial infections are asymptomatic [165] [166] [167], the

potential to develop genital tract sequelae remains unchanged when compared to

individuals that experience symptoms such as postcoital bleeding, fever, abdominal pain,

and mucopurulent vaginal discharge [168]. This is of particular concern given the

reported rate of chlamydial infection among women is over two and half times greater

than the reported rates for men [147] and C. trachomatis infection can ascend into the

female upper genital tract causing long term reproductive complications such as ectopic

pregnancy, involuntary infertility, miscarriage, and increased risk of coinfection and

transmission of other pathogenic agents [169] [170] [171] [172]. As a result, in the

absence of a protective chlamydial vaccine, Chlamydia prevention efforts mainly focus

on increasing the rate of screening and treating infected individuals [134] [170] [173].

While current antibiotic regimens are highly effective in clearing the organism

during uncomplicated lower genital tract infection [174] [175], it has been reported that

doxycycline treatment prevents the development of host immune responses in a murine

model of chlamydial genital infection [162]. Furthermore, Brunham and Rekart

hypothesized that populations were susceptible to reinfection due to a shortened duration

of C. trachomatis encounters which may limit natural immunity [132] [133]. Indeed,

repeated chlamydial exposure has been identified as a risk factor for worse disease in

animal and human studies when compared to reference groups that have only been

exposed once or are considered low-risk sexually transmitted disease populations [176]

[177] [178]. Among a high- risk population of sex workers, reoccurring C. trachomatis

infection was associated with Pelvic Inflammatory Disease [179]. Similarly, in a

retrospective study of 11,000 Wisconsin women, ≥3 positive chlamydial tests increased

the chances of Pelvic Inflammatory Disease diagnosis by a factor of six [177]. Although

these studies indicate an increased risk for severe disease following multiple positive

Chlamydia tests, they fail to distinguish between prolonged exposure and reinfection, or

the role antibiotic intervention played, if any, in the development of disease. This is

further complicated by the initial decline in reported chlamydial infection rates following

the introduction of screening and treatment control programs which have since rebounded

to levels comparable or exceeding those observed prior to the implementation of

intervention strategies.

In this study, we sought to determine if data derived from population surveillance,

human studies, and in vivo experiments could be recapitulated using the urogenital mouse

model as a framework to determine how treatment at various time-points throughout the

course of an active primary infection affects disease outcomes during repeated infection

cycles.

21

Materials and Methods

Mice

7 to 8 week old C57BL/6J mice were purchased from the Jackson Laboratory

(Bar Harbor, ME) and maintained at our facility, which is fully accredited by the

Association for Accreditation and Assessment of Laboratory Animal Care International.

The Animal Care and Use Committee at the University of Tennessee Health Science

Center (Memphis, TN) approved all animal studies.

Chlamydia Strain and Titration

The Chlamydia muridarum Weiss strain was propagated in HeLa 229 cells and

stored at -80oC. Chlamydial titer was evaluated by infecting HeLa 229 cells and counting

Inclusion Forming Units (IFUs) by fluorescence microscopy as previously described

[113].

Mouse Infection

Mice were subcutaneously treated with 2.5 mg of medroxyprogesterone acetate

(Sicor or Depo-Provera, Pfizer, New York, NY) in 0.1 mL of PBS at fourteen and seven

days prior to vaginal infection. Mice were infected by vaginal inoculation with 2 x 103

IFU of C. muridarum in 10 uL of sucrose phosphate glutamic acid (SPG) buffer. For

rechallenge experiments, mice were treated with progesterone and vaginally challenged,

as previously described, fifty-six days post primary infection unless otherwise specified.

Antibiotic Treatment

Mice were treated daily with 0.3 mg of doxycycline hyclate (APP

Pharmaceuticals, Schaumburg, Il) diluted in distilled, endotoxin-free water (HyClone,

Logan, Ut) by interperitoneal injection during the primary infection course only. All

treatment groups received antibiotic for fourteen days consecutively except for

uninfected, negative control mice treated with progesterone. Treatment groups included

treatment from 0-14 d, 14-28 d, 28-42 d, 35-49 d, and Untreated-(UN).

Bacterial Shedding

Vaginal swabs were taken on interval days throughout infection and collected in

2-mL microcentrifuge tubes that contained 0.5 mL of SPG (with three 4mm diameter

round glass beads) and stored at -80oC. Swab samples were thawed and vortexed.

Infectivity was quantified by inoculating Hela 229 monolayers seeded in 48-well plates

with swab supernatant fluid. After 44 h incubation at 37oC in an atmosphere of 5% CO2,

22

plates were washed with PBS-Azide and methanol fixed. Chlamydial inclusions were

visualized and counted by fluorescence microscopy.

Fluorescence-Linked Immunosorbent Assay (FLISA) Antibody Analysis

For total immunoglobulin G (IgG) and IgG isotype studies, HeLa 229 cells were

seeded in 48-well plates (CoStar, Corning, NY) at a cell number of 105 cells/well

overnight. HeLa 229 monolayers were infected with C. muridarum at a multiplicity of

infection (MOI) of two for 2 hours with consistent rocking at 37oC. The innoculum was

immediately aspirated and plates were incubated in Dulbecco’s Modified Eagle Medium

(DMEM) (Life Technologies, Carlsbad, California) complete containing 2ug/mL of

cycloheximide for 36 hours at 37oC in an atmosphere of 5% CO2. Following incubation,

plates were washed 3 times with PBS-Azide and fixed with 500 uL of methanol per well

for 10 minutes. After fixation, plates were washed 3 times with PBS-Azide and incubated

at 37oC with a 1:500 dilution of murine sera, pooled based on antibiotic treatment group,

in PBS/0.1% grade IV ovalbumin/0.05% Tween 20. After incubation, plates were washed

3 times and incubated with FitC-conjugated rat anti-mouse IgG, IgG1, IgG2a, IgG2b

(Southern Biotech, Birmingham, AL), FitC-conjugated goat anti-mouse IgG2c, or Alexa

Fluor ® 488 -conjugated goat anti-mouse IgG3 (Invitrogen by Life Technologies,

Carlsbad, California) for thirty minutes. Plates were washed 3 times with PBS-Azide and

fluorescence intensity was immediately read as fluorescent light units (FLUs) at

sensitivity 70 using a Biotex Synergy 2 Microplate Reader. Results represent the mean of

triplicate values for each pooled sample group while the solid horizontal bar denotes

background intensity as measured by normal sera from uninfected untreated animals.

Pathology

Immediately following sacrifice, individual mice were grossly examined for

genital tract pathology abnormalities and photographs were taken. The uterine horns and

oviducts were collectively and separately scored based on dilation and visual

inflammation (reddening and blockage) of the aseptically removed urogenital tissue.

Scoring for dilation of uterine horn and oviduct tissues was as follows: 0, normal/no

significant dilation; 1, mild dilation of a single cross-section; 2, one to three dilated cross-

sections; 3, more than three dilated cross-sections; 4, confluent dilation. Excised tissues

were methanol fixed and paraffin-embedded. Longitudinal sections were later stained

with one of the following: hematoxylin and eosin (H&E), NIMP-R14 (neutrophil

identifying marker), CD68 (macrophage identifying marker), CD4 (T-cell identifying

marker), and Toluidine Blue (mast cell identifying stain).

Organ-Total Body Weight Ratio Analysis

Mice were individually weighed to obtain the total body weight in grams.

Immediately following sacrifice, the genital tract was aseptically removed and weighed in

23

milligrams. Organ-total body weights were expressed as a function of organ weight

divided by total body weight to generate weight ratios. Weight ratios for each animal

were averaged by corresponding antibiotic treatment group.

Statistical Analysis

Statistical significance of differences between treatment groups was analyzed

with GraphPad Prsm 4 software (La Jolla, CA) using one-way analysis of variance

(ANOVA) with post hoc Tukey testing comparing all possible pairs of means unless

otherwise noted. The strength of linear correlations was determined using Pearson

product moment correlation coefficient unless otherwise noted. All analyses were

performed using two-tailed testing at a 95% confidence interval of difference.

Results

Kinetics of Genital Tract Infection in Antibiotic-Treated Mice

To evaluate the effect of antibiotic treatment on the duration of chlamydial

infection, cervicovaginal swabs were collected throughout the primary course of

infection. Progesterone treated mice were vaginally infected with 2 x 103 IFU

chlamydiae, treated with doxycycline and swabbed as described in Materials and

Methods. Mice infected but not treated displayed kinetics of an active infection shedding

3-5 log10 of viable organisms within the first 3 weeks (Figure 2-1). These untreated

animals were culture negative, indicating a natural resolution of lower genital tract

infection, by approximately week four. We were unable to recover infectious organisms

at day four after inoculation in early treated animals, the earliest time point analyzed.

This suggests that antibiotic treatment prevented intravaginal chlamydial colonization,

negating the establishment of an active urogenital tract infection. Mice that started

treatment at an intermediate time point (14-28 d) exhibited a rapid reduction in

recoverable organisms after the onset of treatment, whereas, groups treated late during

the infection cycle (treatment initiation at day 28 or 35) paralleled untreated clearance

rates.

Immune Arrest and Pathology Following Primary Infection of Antibiotic-Treated

Mice

Antibiotic treatment has been shown to prevent the onset of host immune

responses and the development of upper tract pathology post primary infection [162]

[180]. To assess whether an immunological response had occurred we measured systemic

chlamydial specific IgG antibody titers post primary infection by FLISA in addition to

scoring the severity of disease. As hypothesized, pathology following primary challenge

was significantly reduced in early treatment groups when compared to later treatment

groups or untreated controls (Figure 2-2). Animals that began treatment at 14 days post

24

Figure 2-1. Shedding of viable C. muridarum following intravaginal challenge.

Note: Solid lines represent various antibiotic treatment groups. Blue: Untreated control,

Red: (0-14 d), Green: (14-28 d), Purple: (28-42 d), Aqua: (35-49 d). Symbols represent

the mean inclusion forming units (IFUs) per milliliter recovered at each time point. Error

bars denote standard deviation (SD) on a log10 scale.

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

0 4 8 16 28 35

Ce

rvic

o-v

agin

al s

he

dd

ing

IFU

/mL

Time (Days Post Infection)

UN

(0-14)

(14-28)

(28-42)

(35-49)

25

Figure 2-2. C57BL/6 pathology severity scores 49 days post-primary C.

muridarum intravaginal infection.

Note: Solid bars represent the mean severity score for each treatment group. Symbols

represent individual animals. Differences between treatment groups were observed by

one-way analysis of variance (ANOVA) with post-hoc analysis using a Tukey-Kramer

Multiple Comparison test. * denotes p < 0.05 at 95% confidence interval of difference. **

denotes p < 0.01 at 95% confidence interval of difference. No difference was observed

between untreated animals and animals treated at 14-28d, 28-42d, and 35-49d. These data

are representative of multiple experiments.

Untr

eated

(0-1

4)

(14-

28)

(28-

42)

(35-

49)

-1

0

1

2

3

4

5

***

*

Doxycycline Treatment

Sev

erit

y S

core

26

primary infection displayed reduced disease scores (mean of 1, mild disease) when

compared to moderately diseased untreated animals and late treatment groups. In

addition, the severity of upper genital tract complications followed the same trend

established with the development of chlamydiae-specific immune responses (Figure 2-3).

In treatment groups where Chlamydia- specific antibody titers were observed as

determined by FLISA analysis, chlamydiae-related genital pathology was more severe.

Total anti-chlamydial IgG antibody titer was undetectable in early treated animals and

was significantly different in comparison to untreated animals and animals treated at 14-

28d, 28-42d, and 35-49d. Anti-chlamydial IgG levels resembling those of untreated

controls suggested a role for immune-mediated upper tract pathology in the development

of adverse disease outcomes.

Given the importance of anti-chlamydial Th1-dominant adaptive responses in the

resolution of chlamydiae genital infection [181] [182] [183] [184], we evaluated the

effects of antibiotic intervention on isotype switching as a marker for Th1 verses Th2

biased responses in a FLISA assay with C. muridarum-infected HeLa 229 cells as

antigen. IgG isotypes were undetectable following early treatment but data for untreated

and late treated animals strongly suggested a Th1 bias, evidenced by IgG2 dominance,

regardless of the onset of antibiotic intervention (Figure 2-4). Collectively, these data

implicate a bias for Th1 immunity irrespective of severe disease development or the

initiation of anti-chlamydial drug intervention.

Disease Severity Following Reinfection in Antibiotic-Treated Mice

Repeated infections are common in women and early treatment and have been

postulated to increase reinfection susceptibility on the population level [163] [185] [186].

Therefore, it was important to assess whether immune arrest during primary infection

contributed to disease severity post-secondary infection in our model. As a result, mice

were rechallenged 56 days after primary vaginal encounter to assess disease severity

following repeated infection. The animal groups are reflective of the onset of antibiotic

treatment during primary infection as animals did not receive treatment during the

secondary infection course. Gross examination of early treatment samples showed

significant differences in the magnitude and localization of Chlamydia-induced

pathology. Data presented in (Figure 2-5) show that reinfected animals treated at day

zero during primary infection had a mean disease severity score of 1.6, indicating mild to

no disease. Conversely, animals treated at days fourteen, twenty-eight, and thirty-five

displayed moderate to severe disease, with mean severity scores ranging from 2-3.1. To

ascertain differences in the localization of disease, we separately scored uterine horn

(Figure 2-6A) and oviduct (Figure 2-6B) pathology. Early treatment resulted in

significantly less endometrial disease when compared to untreated controls or animals

treated at fourteen, twenty-eight, and thirty-five days post primary infection. Gross

evaluation of the oviduct pathology revealed mild disease irrespective of the onset of

treatment.

In contrast to human isolates, C. muridarum may result in pathology and

27

Figure 2-3. Total Immunoglobulin G (IgG) antibody response 49 days post-

primary C. muridarum intravaginal infection.

Note: Bars represent mean fluorescent light units (FLUs) for each treatment group.

Differences between treatment groups were observed by one-way analysis of variance

(ANOVA) with post-hoc analysis using a Tukey-Kramer Multiple Comparison test. **

denotes p = 0.001 at 95% confidence interval of difference.

Nor

mal

Buf

fer

Unt

reat

ed

(0-1

4)

(14-

28)

(28-

42)

(35-

49)

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000 p=0.001**

Doxycycline Treatment

Mean

FL

U

28

Figure 2-4. Immunoglobulin G (IgG) isotype responses 49 days post C.

muridarum intravaginal infection in mice intraperitoneally treated with doxycycline.

29

Figure 2-5. Incidence and severity of upper genital tract sequelae in antibiotic-

treated mice 28 days post-secondary infection.

Note: Solid bars represent the mean severity score for each treatment group. Symbols

represent individual animals. Data depicted here represents trends seen in multiple

experiments.

30

Figure 2-6. Incidence and severity of uterine horn and oviduct sequelae in

antibiotic-treated mice 28 days post-secondary infection.

Note: Solid bars represent the mean severity score for each treatment group. Symbols

represent individual animals. Data depicted here represents trends seen in multiple

experiments.

31

infertility after a single infection in mice [187]. Consequently, we questioned whether the

the increase in disease severity following secondary infection was a result of primary

disease worsening over time or if repeated infection compounded sequelae severity. To

determine whether secondary challenge resulted in more severe disease when compared

to disease post primary infection, we compared weight ratios as a measure of objective

disease severity for three independent untreated groups as weight positively correlated

with disease severity score (Figure 2-7). As depicted in (Figure 2-8), untreated C.

muridarum challenged animals were sacrificed fifty-six days post primary infection

(Prim56) and were compared to untreated C.muridarum challenged mice sacrificed one

hundred and twelve days post primary infection (Prim112). Prim56 and Prim112 mg/g

ratio means remained stable despite a fifty-six day gap between sacrifice dates. Albeit not

statistically significant, untreated C. muridarum challenged animals sacrificed fifty-six

days post-secondary infection (Sec56), displayed an increase in mg/g ratio when

compared to animals that only received primary infection. These data suggest severe

disease is compounded with repeated chlamydial exposure as opposed to residual primary

disease progressively worsening over time.

IL-4 Is Detectable Late Following Reinfection and Positively Correlates with Disease

Severity in Antibiotic-Treated Mice

Little is known about the cytokine milieu in the late stages of recurrent

chlamydial infection. To address the question of which, if any, cytokines were detectable

systemically during reinfection cycles, IFN-gamma, TNF-alpha, GM-CSF, IL-1B, IL-2,

IL-4, IL-6, IL-8, IL-10, and IL-12 were simultaneously analyzed from individual sera

samples collected from mice sacrificed 56 days post-secondary infection. Our results

implicate a relatively short-lived TH1 response as suggested by a dominant IgG2 antibody

response (Figure 2-2) and unremarkable detection of IFN-gamma, TNF-alpha, GM-CSF,

IL-1B, IL-2, and IL-12 post recurrent infection (See appendices). Interestingly, IL-4, a

potent inducer of TH2 immunity and a key defensive player against extracellular

pathogens, was detected in the circulation and positively correlated with an objective

measure of disease severity, weight ratios (Figure 2-9). Moreover, IL-4 detection in the

observed sera analytes gradually increased with the deferred treatment of primary

infection suggesting a TH2-independent role for IL-4 in this model.

Chlamydia and Cellular Infiltrates Following Reinfection in Antibiotic-Treated Mice

Genital tract sections were stained with H&E, NIMP-R14, CD68, CD4, or

Toluidine to identify abundant cell types present in the genital tract after recurrent

infection, including potential populations of known IL-4 secreting cells. Late treated

tissues stained with neutrophil-specific marker, NIMP-R14, revealed dense

polymorphonuclear populations when compared to the residential populations observed

in uninfected, untreated controls. Tissues excised from early treated animals

(Figure 2-10B) displayed far fewer neutrophils than late treated groups (Figure 2-10A).

Levels of T-cells expressing CD4 and macrophages expressing CD68 were comparable to

32

Figure 2-7. Weight ratio verses total severity score correlative analysis.

Note: Pearson’s correlation was used to determine whether a relationship existed between

severity scoring and weight ratio variables. Solid line represents the line of best fit. Blue

circles represent individual mice (N=30). Red box highlights a significant positive

correlation between the two variables, weight ratios and total disease severity, using a

two-tailed analysis at 95% confidence interval of difference.

0 5 10 15 200

1

2

3

4

5

Pearson r

95% confidence interval

P value (two-tailed)

P value summary

Is the correlation significant? (alpha=0.05)

0.5105

0.1840 to 0.7355

0.0039

**

Yes

Weight(mg/g)

Severi

ty S

co

re

33

Figure 2-8. Comparative analysis of weight ratios post-primary and secondary

sacrifice in untreated intravaginally infected C57BL/6 mice.

Note: Disease severity was assessed by dividing individual genital tract weights (in

milligrams) by the corresponding total weight of the animal (grams) as described in

Materials and Methods. Bars represent the mean weight ratio from three independent

studies for each infection cycle/time group while error bars correspond to the standard

error of the mean (SEM). Prim56 animals (N=3) were sacrificed 56 days post primary

infection. Prim 112 animals (N=8) were sacrificed 112 days post-primary infection.

Sec30 animals (N=9) were sacrificed 56 days post-secondary infection, totaling 112 days

post initial chlamydial exposure.

Prim56 Prim112 Sec560.0

2.5

5.0

7.5

10.0

12.5

15.0

Infection Cycle/Time (Days)Post Infection

Weig

ht

Rati

o

34

Figure 2-9. Systemic IL-4 post recurrent infection.

Note: Sera collected by submandibular bleed post-secondary infection was subjected to

Luminex 10-Plex analysis to determine the cytokine milieu during late stages of recurrent

infection. Individual samples were plated in duplicate and bars represent the mean IL-4

concentration for each group (Panel A). To determine whether IL-4 correlated with

disease, Pearson’s correlation was used (Panel B). A significant positive correlation (p=

0.0462) was observed when IL-4 was plotted against the weight ratios of corresponding

animals (N=30). Solid black line represents the line of best fit.

35

Figure 2-10. Neutrophil infiltrates post-secondary infection.

Note: Immunohistochemistry evaluation using NIMP-R14, a neutrophil-specific marker,

of genital tract samples taken 56 days post-secondary C. muridarum infection revealed

marked increases in neutrophil influx in untreated controls (Panel A, x20) when

compared to early treatment groups (Panel B, x20) and normal, non-infected tissue (Panel

C, x20). Representative samples from each group are shown.

A B

C

36

levels observed in untreated, uninfected controls (data not shown) assessed at the same

time-point.

As previously mentioned, we detected unexpected concentrations of systemic IL-

4 (Figure 2-9) despite seeing the antibody surrogate marker for TH1-related cytokines,

IgG2 (Figure 2-4). As a result we sought to histologically identify known IL-4 producing

cell populations. Using Toluidine Blue stain we identified a treatment dependent increase

in genital tract mast cell populations, the number of mast cells increased the longer

treatment was deferred (Figure 2-11). Furthermore, there was a significant correlation

(p= 0.0090) between the severity of disease as assessed by weight ratios and mast cell

density (Figure 2-12). We also identified eosinophil infiltrates in H&E stained genital

tissue preparations but no statistically significant association was observed (Figure 2-13).

Discussion

In this study, the main objective was to integrate various Chlamydia observations

into a single, comprehensive mouse model of chlamydial genital infection. The

reproductive health of women is of particular concern with current CDC Chlamydia

surveillance data indicating increases in reported chlamydial infections, perhaps, in part,

as a result of rapid antibiotic intervention. In contrast, rising infection rates are coupled

with reduced complication rates in screened populations [131] [134] [188] despite

evidence of adverse complications being linked to multiple or prolonged chlamydial

exposures in human epidemiological studies [176] [177] [179]. Collectively, these

observations underscore the importance of determining the impact of antibiotic

intervention on the development of severe upper tract disease following repeated

infection.

In this C. muridarum model of genital infection, we showed that mice

intravaginally infected and treated early during primary infection displayed rapid

bacterial clearance kinetics in the lower genital tract, significantly reduced anti-

chlamydial IgG titer, and virtually no upper genital tract disease. These results verify and

validate a previous antibiotic study in C57BL/10 mice that concluded early antibiotic

treatment reduced the development of natural immunity [162]. Our study expands on this

model by extending observation on disease severity to post-secondary infection and by

defining differences in disease localization. Mice treated early during primary infection

displayed mild disease severity scores (mean score of 1) in both uterine horn dilation and

hydrosalpinx formation. Conversely, untreated infected mice and mice treated later

during primary infection displayed moderate to severe disease severity scores in the

uterine horn (mean range of 2 to 3.3). However, in the oviduct, significant differences

were not observed between treatment groups suggesting either chlamydiae-related

responses differ between these two tissues or severe endometrial disease limits the

likelihood of oviduct disease. Interestingly, and perhaps not logically, mice that received

treatment midway through the natural course of an active primary infection (14-28d)

displayed elevated disease severity scores in both the uterine horn and the oviduct, albeit

not significantly different from other treatment groups. So far, we have not studied this

phenomenon extensively but eradication of the organism soon after the initiation of

37

Figure 2-11. Mast cell infiltrate post-secondary infection.

Note: Immunohistochemistry evaluation using Toluidine Blue, a mast cell identifying

stain, of genital tract samples taken 56 days post-secondary C. muridarum infection

revealed mast cells in all of the groups assessed. Organ specific mast cell density was

determined by manually counting positively stained granulated mast cells in the genital

tissue. Bars represent mean mast cell counts for each treatment group.

38

Figure 2-12. Mast cell infiltrate verses systemic IL-4 correlative analysis.

Note: Organ specific mast cell density was determined by manually counting positively

stained granulated mast cells in the genital tissue. A strong positive correlation between

systemic IL-4 and mast cell infiltrate (r= 0.6682, p= 0.0090) was observed using

Pearson’s correlation two-tailed testing at a 95% confidence interval.

39

Figure 2-13. Eosinophil infiltrate post-secondary infection.

Note: Immunohistochemistry H&E staining of genital tract samples taken from mice 56

days post-secondary infection with C. muridarum revealed eosinophils in untreated,

uninfected animals (A, x40), early treatment groups (B, x20), and late treatment groups

(C, x30). Thirty-six fields were randomly selected and counted (12 cervical fields, 12

uterine horn fields, and 12 oviduct fields.) for each sample. No significant correlation

between the number of eosinophils present in the genital tract and systemic IL-4 was

found (D). Representative samples from each group are shown.

40

antibiotic treatment and detectable humoral antibody responses fail to explain the

observation. Future characterization of the model, including but not limited to the cellular

immune response, is of interest and should be investigated to elucidate the underlying

immune mechanism involved.

Histological analyses of various cellular populations suggest an influx of

polymorphonuclear cells during late stages of recurrent urogenital infection. Using

NIMP-R14, a neutrophil –specific marker, we observed an increase in neutrophils among

late treated animals when compared to uninfected controls and early treated groups. This

follows previous observations by our group [189] [190] and others [191] [192] [193] that

cellular infiltrates composed of neutrophils correlate with the development of more

severe disease. Data from our studies illustrate the sustained presence of neutrophils

during late stages of recurrent infection (fifty six days post-secondary infection) and

suggest that disease severity may be a function of sustained innate reactivity rather than

the TH1-biased response that was observed in all animals, irrespective of disease severity.

This expands the current paradigm of neutrophil influx as an early marker of infection

into a potential mediator of disease outcomes and implies that modulation of neutrophil

influx may improve chlamydiae-related disease severity and may be investigated as a

potential way to improve reproductive health.

Perhaps the most interesting finding in this investigation was the systemic

detection of TH2-linked immune regulator, IL-4 (Figure 2-9), with greater detection of

IL-4 corresponding with late treatment groups displaying the most severe disease. While

our data support the current dogma of TH1-type responses playing a role in the resolution

of infection as evidenced by a detectable IgG2-biased chlamydial-specific antibody

response (Figure 2-4), they also suggest an alternative TH2 independent role for IL-4

production during recurrent chlamydiae infection. Our data illustrate the development of

chlamydial-induced genital tract pathology, with the severity of disease increasing the

longer you defer antichlamydial treatment and with multiple rounds of chlamydial

exposure. Following secondary exposure, a scenario in which we report exacerbated

genital tissue damage, large amounts of IL-4 were detectable and gradually increased as

the magnitude of disease increased. Our data compliments observations made by Holland

et al of increased TH2-related cytokine production in patients with more severe disease

[194]. Miquel et al reported that women with a history of Chlamydia trachomatis

infection secrete cytokines consistent with TH2 immunity, specifically significant levels

of IL-4, in response to ex vivo stimulation [195]. Collectively, these observations lead us

to hypothesis a role for IL-4 in tissue repair. Recent reports have linked IL-4 to tissue

repair and wound healing by alternatively activating macrophages and stimulating the

expansion of IL-4 producing cells [196] [197] [198] [199] [200]. In line with these

reports, we were able to detect both eosinophils and mast cells, known IL-4 producing

cells, in our model. Furthermore, it is important to keep in mind that we detected a TH1

response, at least early on during infection, as evidenced by an IgG2 biased antibody

response. This suggests that the production of IL-4 occurs later in the immunological

cascade (following the production and secretion of chlamydial-specific antibody), which

decreases the likelihood of IL-4 initiating the genital tract tissue damage observed (the

41

idea of IL-4 being an injury promoter) and points to the possibility of IL-4 playing a role

in “injury response.”

While we present data in support of the IL-4-driven injury response hypothesis,

further study is necessary for hypothesis validation and mechanism delineation. For

instance, we proposed the transient production of TH1-related cytokines led to the

detection of IgG2 isotypes during early infection cycles. Time course studies following

the onset and dissipation of TH1-linked cytokines and IL-4 are key to verifying that

proposal. While we know eosinophils and mast cells are capable of producing IL-4 and

their presence in the genital tract implicates them as IL-4 sources, it would be useful to

know if they are actually responsible for producing the detected IL-4. This can be

accomplished by microarray analysis of IL-4 gene expression and subsequent ELISA

assay for IL-4 production ex vivo. Additional experiments may also include looking at

the upregulation of genes known to particulate in repair signaling pathways in genital

tract-derived tissue and dispelling the possibility of IL-4 participating in the initial stages

of disease development using IL-4-/- knockout mice.

Rapid antibiotic treatment is a key component of current Chlamydia control

initiatives that may influence infection rates and, more importantly, disease outcome

trends on the population level, but assessing the impact of antibiotic intervention on

severe disease development is difficult to address due to ethical concerns associated with

delaying or withholding treatment in the human population. To this end, we interrogated

the murine model for its potential to serve as a translational bridge between population-

based epidemiology and previous animal and human studies. Our results reaffirm the

benefit of rapid antibiotic intervention in Chlamydia control efforts and suggest an added

benefit during repeated infection even in the absence of antibiotic treatment. Although

early-treated mice developed some upper tract disease after re-challenge, it was

substantially less severe than the disease observed in groups where treatment was

delayed. This is of particular interest since previously screened and treated women

commonly re-enter susceptible sexual networks, acquire a secondary infection, and fail to

seek treatment due to the asymptomatic nature of Chlamydia trachomatis infection. Most

importantly, we provide support for IL-4 dependent tissue damage response following the

induction of chlamydial-induced genital tract complications. We conclude that the

integrated murine model is useful for studying the efficacy of antibiotic intervention at

varying time-points with respect to disease severity but requires further characterization

to elucidate potential mechanisms by which chlamydial pathogenesis occurs and is

regulated, particularly as it pertains to the induction of IL-4. The dichotomy between cell-

mediated chlamydiae-related immune responses may not be as straightforward as we

previously thought.

42

CHAPTER 3. CHLAMYDIAL GENITAL TRACT SEQUELAE IS AGE AND

STRAIN-DEPENDENT

Introduction

Sexually transmitted Chlamydia trachomatis is the causative agent of the most

commonly reportable disease in the United States [147] and a small percentage of

infected women develop reproductive and gynecologic complications such as chronic

pelvic pain [170, 201-204], pelvic inflammatory disease (PID)[170] [201] [205] [206],

involuntary infertility [170] [172] [201] [203] [206], and ectopic pregnancy [170] [172]

[176] [201] [204] [206]. While many factors contribute to individual disease variability to

chlamydiae [74] [75] [100] [207], the effects of biological determinants such as genetic

predisposition and host age at the time of infection [208] [209] are largely unknown.

Identification of host genetic factors is central to improving the quality of current disease

animal models and understanding chlamydial-induced disease heterogeneity. Moreover,

murine genetic studies may implicate novel pathway targets for individualized

therapeutic intervention in humans.

Inbred mouse models have been extensively used to study host contributions to

chlamydiae infection susceptibility [74] [75] [210] [211] [212] [213] [214] [215].

Cytokine induction and expression, H2 haplotypes, chlamydial species, and Th-1-

mediated responses have all been identified as factors that contribute to varying murine

susceptibility. More recently, unbiased genome-wide approaches have been used to

identify p47 GTPase genes encoded on chromosome 11 that confer resistance in mice to

systemic models of C. trachomatis and C. psittaci infection, respectively [190] [216]

[217]. Since, Miyairi et al have combined the high throughput forward genetic approach

of BxD recombinant inbred strains with Bayesian network modeling to identify host

pathways that modulate susceptibility to systemic chlamydial infection [78].

In the present investigation, we considered the age of the animal at time of

infection and the genetic composition of the host when identifying differences in upper

genital tract disease severity. We conclude that age and host genetic make-up are both

important determinants of disease.

Materials and Methods

Mice

Seven week old C57BL/6J (H-2b) and DBA/2J (H-2d) female mice (referred to as

≤9wk animals) were commissioned from The Jackson Laboratory (Bar Harbor, ME,

USA). Fourteen to 32-week old C57BL/6J and DBA/2J female mice (referred to as

>14wks) were bred and maintained at our facility, which is fully accredited by the

Association for Accreditation and Assessment of Laboratory Animal Care International.

43

The Animal Care and Use Committee at The University of Tennessee Health Science

Center (Memphis, TN) approved all animal studies.

Chlamydia Strain

The Chlamydia muridarum (Weiss) strain was propagated in HeLa 229 cells and

elementary bodies (EBs) were purified from infected cells by 30% density gradient

centrifugation in Renograffin (E.R. Squibb and Sons, Princeton, NJ). Chlamydial titer

was evaluated by infecting HeLa 229 cells and enumerating by indirect fluorescence

microscopy as previously described. Stocks were stored at -80oC until use.

Chlamydia muridarum Infection

DBA/2J, C57BL/6J, and BXD mice were pretreated with progesterone to

synchronize the estrous cycle and stabilize the target genital epithelium. Pretreatment

consisted of two subcutaneous injections of 2.5 mg medroxyprogesterone acetate

injectable suspension (Sicor, Irvine, CA) in 0.1 mL phosphate buffered saline (PBS),

given at 10 and 3 days prior to infection. Mice were inoculated with 5000 inclusion-

forming units (IFUs) contained in 5 uL sucrose phosphate-buffered glutamic acid (SPG)

via the vaginal vault with a filtered micropipettor on day 0. Five animals from each group

(DBA/2J-≤9wks, DBA/2J->14wks, C57BL/6J-≤9wks, and C57BL/6-2J->14wks) were

sacrificed 40-days post infection. For rechallenge studies, five animals from each group

were progesterone treated at day 30 and intravaginally challenged with 5000 IFU at day

40. All rechallenged animals were euthanized 30 days post reinfection, seventy days post-

primary infection.

Pathology Assessment

Individual mice were weighed to obtain total body weight in grams. In situ

macroscopic examinations were performed for evidence of upper genital tract

abnormalities immediately following sacrifice. Genital tissues were then aseptically

removed, photographed, and weighed to obtain organ weight in micrograms. Severity

scores were determined for the uterine horn and oviducts separately: 0, no significant

dilatation; 1, mild dilatation of a single cross-section; 2, one to three dilated cross-

sections; 3, greater than three dilated cross-sections; 4, confluent pronounced dilation.

Scores assigned to individual mice were averaged to obtain the mean severity score for

each group of animals. Weight ratios (organ weight-milligrams/ total body weight-grams)

were used as an objective validation tool for upper genital tract severity scoring.

44

Results

C57BL/6J Exhibit Age-Dependent Upper Genital Tract Sequelae Post-Primary

Intravaginal Infection

DBA/2 and C57BL/6 female animals of varying ages were intravaginally infected

with C. muridarum as described in materials and methods. Individual mice were scored

according to the localization of disease 40 days post infection. Nine out of 20 animals

(45%) assessed developed hydrosalpinx with B6-≤ 9wk animals displaying significantly

more oviduct disease (p< 0.001) when compared to all other animal groups (Figure

3-1A). Twenty percent of DBA/2, regardless of age, displayed mild oviduct dilatation

indicated by a mean severity score of 0.23. Conversely, discordant oviduct severity

scores were observed among C57BL/6J-≤ 9wk and C57BL/6J->14wk groups. A

moderate oviduct severity score of 2.2 was observed in B6-≤ 9wk animals while B6-

>14wk animals displayed mild oviduct involvement with a mean score of 0.4. No

significant differences were observed in endometrial dilatation among groups,

irrespective of inbred strain or age (Figure 3-1B).

Secondary Intravaginal Infection Exacerbates Chlamydial-Induced Upper Genital

Tract Complications

We hypothesized that recurrent chlamydial exposures would result in worse

disease when compared to animals that received primary infection alone. To test this

hypothesis, DBA and B6 female mice were intravaginally infected at day 0 and

rechallenged at day 40 as described in materials and methods. Thirty days after

rechallenge, genital tissues were aseptically harvested and assessed for oviduct and

uterine horn dilatation. As we hypothesized, secondary disease exacerbated negative

disease outcomes in both strains and age groups (Figure 3-2) when compared to the

corresponding groups that only received a primary infection (Figure 3-1). Sixteen out of

21 animals (76%) examined after secondary infection developed oviduct pathology as

opposed to 45% of primarily infected animals. Fourteen out of 21 animals (66.6%)

examined after secondary infection developed uterine horn pathology as opposed to 30%

of primarily infected animals.

C57BL/6 Mice Are More Susceptible to Chlamydial-Induced Upper Genital Tract

Complications When Compared to DBA/2J Mice

DBA and B6 female mice were intravaginally infected at day 0 and rechallenged

at day 40 as described in materials and methods. Thirty days after rechallenge, genital

tissues were aseptically harvested and assessed for oviduct and uterine horn dilatation.

Oviduct disease observed in C57BL/6-≤ 9wk animals was significantly worse (mean

severity score 3.6) than that observed in DBA/2-≤ 9wk and DBA/2->14wk animals with

mean scores of 0.8 and 1.2, respectively. Additively, C57BL/6->14wk animals displayed

significantly more severe oviduct disease than that seen in DBA/2-≤ 9wk or DBA/2

45

Figure 3-1. Upper genital tract disease incidence and severity post-primary

infection.

Note: DBA/2 and C57BL/6J female mice were intravaginally infected with Chlamydia

muridarum as described in materials and methods. Forty days after infection, mice were

sacrificed and pathology was assessed grossly. The left figure (Panel A) displays oviduct

disease severity and disease incidence by inbred strain and age group. The right figure

(Panel B) displays uterine horn disease severity and disease incidence by inbred strain

and age group. 5 animals were assessed per group and bars indicate mean severity scores.

* and ** indicate significant differences (p≤ 0.01 and p≤ 0.001) as determined by one-

way ANOVA with post hoc Newman-Keuls Multiple Comparison Test. Data are

representative of two independent studies.

46

Figure 3-2. Upper genital tract disease incidence and severity post-secondary

infection.

Note: DBA/2 and C57BL6 female mice were intravaginally infected and rechallenged

with Chlamydia muridarum as described in materials and methods. Thirty days post-

secondary infection, mice were sacrificed and pathology was assessed grossly. The left

figure (Panel A) displays oviduct disease severity and incidence by inbred strain and age

group. The right figure (Panel B) displays uterine horn disease severity and incidence by

inbred strain and age group. 5-6 animals were assessed per group and bars indicate mean

severity scores. * indicate significant differences (p<0.01) as determined by one-way

ANOVA with post hoc Newman-Keuls Multiple Comparison Test. Data are

representative of two independent studies.

47

->14wk animals. Although uterine horn severity assessments did not result in statistically

significant differences, B6-≤ 9wk animals consistently displayed moderate disease

severity as indicated by a mean score of 2 while B6->14wk endometrial involvement was

comparable to that observed in DBA/2 animals.

To remove any unintentional disease bias imposed by using the quantitative

severity scoring system, we assessed disease severity as a function of organ weight in

milligrams divided by total body weight in grams (Figure 3-3). We have previously

observed a direct correlation between severe disease and increased weight ratios given

genital abnormalities that involve dilatation of murine oviduct and uterine horn tissues

increase the ratio numerator value-organ weight in milligrams (Figure 2-7). Weight

ratios compliment severity score data in that secondary infection results in worse disease

when compared to animals of the same strain and age group. Moreover, C57BL/6-≤ 9wk

animals are most susceptible to chlamydial-induced upper genital tract complications

evidenced by significant differences observed in recurrent infection oviduct severity

scores and objective weight ratio analysis.

Discussion

Individual differences in disease susceptibility as a result of infectious agents

exist in the population. For example only a small subset of women with uncomplicated

chlamydial genital infection go on to develop severe upper genital tract

complications[170]. Furthermore, adolescent females are more likely to contract

Chlamydia than any reported age group [185] [218] [219]. In the United States alone, the

reported 2011 rates of sexually transmitted chlamydial infection for women between the

ages of 15-19 and 20-24 were 3416.5 and 3722.5 per 100,000 population, respectively

[147]. The reported rate for adolescents between the ages of 15-19 yrs is over five times

the national rate of 648.9 per 100,000 population and almost six times the national rate

for young women between the ages of 20-24 yrs. These rates are particularly alarming

considering the rate for women in their mid-forties to early fifties is only 35.8 per

100,000 population [147]. Taken together, current epidemiological trends highlight the

need for chlamydial models to account for biological factors such as host age when

assessing incidence and severity of chlamydiae-induced reproductive complications.

In this study, chlamydial-induced disease outcomes were largely dependent on the

mouse age and strain used to model urogenital infection. C57BL/6-≤ 9wk mice were

more susceptible to oviduct pathology when compared to DBA/2 mice as evidenced by

mean severity score post primary (Figure 3-1A) and secondary infection cycles (Figure

3-2A). In addition to extra-strain disease variation, intra-strain disease variation among

B6 was shown to be age-dependent in the oviduct post primary infection and, albeit not

statistically significant, in the oviduct and uterine horn post-secondary infection. In

addition to the four-tiered semi-quantitative severity scoring system, these results were

objectively validated with weight ratio analysis (Figure 3-3). BL/6-≤ 9wk animals

display significantly more disease as evidenced by weight ratios when compared to

48

Figure 3-3. Weight ratio disease severity validation post-primary and secondary

intravaginal infection.

Note: DBA/2 and C57BL6 female mice were intravaginally infected and rechallenged

with Chlamydia muridarum as described in materials and methods. Weight ratios were

determined by dividing individual organ weights (milligrams) by total body weight

(grams). Ratios were averaged to calculate the mean value for each group (indicated by

bar). Error bars depict the standard error of the mean. * indicate significant differences

(p< 0.05) as determined by one-way ANOVA with post hoc Newman-Keuls Multiple

Comparison Test. ** indicate significant differences (p< 0.01) as determined by one-way

ANOVA with post hoc Newman-Keuls Multiple Comparison Test. Data are

representative of two independent studies.

49

> 14wk animals with the same genetic background. Interestingly, disease severity as a

function of weight observed in DBA/2 animals remained consistent irrespective of age or

disease localization.

While inter-strain susceptibility variations have been appreciated for some time

[74] [75] [210] [220], our studies suggest C57BL/6 animals are susceptible to

chlamydial-induced genital tract complications when compared to DBA/2 mice as

determined by semi-quantitative severity scoring (Figures 3-1 and 3-2) and weight ratios

(Figures 3-3). Kaltenboeck et al also found that C57BL/6 animals were susceptible to

chlamydial-induced disease using a respiratory murine model of infection [221]. In their

studies C57BL/6 mice developed severe pneumonia while BALB/C mice were

completely void of symptoms following intranasal priming with C. psittaci and

subsequent challenge. Interestingly, our results and those seen in the Kaltenboeck model

conflict with genital studies proposing a resistant phenotype for C57BL/6 when

compared to C3H and BALB inbred strains, both of which are commonly referred to as

susceptible strains given their increased chlamydial shedding and longer courses of

infection when compared to C57BL/6 mice. Although we have not delineated a

mechanism for the results seen in our study, immunological validation is warranted and

currently underway. The discrepancies observed between our results and those published

by others may also be attributed, at least in part, to differences in the strains of Chlamydia

used, inoculum dosage, murine strains, multiple rounds of infection, and variation in what

is defined as resistance or susceptibility (I. E. reduced shedding, shorter infection course,

infertility, eradication of the infectious agent in the lower and upper genital tract, or the

development of upper genital tract sequelae).

As many researchers developing infectious disease models, we are interested in

identifying human correlates of phenotypic differences seen in our model. Studies are

currently underway using B (C57BL/6) x D (DBA/2J) recombinant inbred mouse strains

to identify genetic factors that may modulate disease susceptibility in an age-dependent

manner. By interrogating the GeneNetwork database using a forward genomic approach,

we hope to identify genes associated with the modulation of oviduct and uterine horn

disease severity in an age and strain dependent manner. More importantly, we are

interested in identifying human correlates for the potential murine-derived phenotypic-

genetic links observed in our model.

While studies aimed at delineating causal mechanisms for the age and strain

dependence reported here are currently underway, these initial observations have

immediate implications for the use of mouse models in understanding chlamydial-

induced disease. Furthermore, these data suggest that the host genetic contribution to

genital pathogenesis may be more complex than previously appreciated.

50

CHAPTER 4. IMMUNIZATION WITH C. MURIDARUM OUTER MEMBRANE

COMPLEX FAILS TO PROTECT AGAINST UPPER GENITAL TRACT

COMPLICATIONS IN A MURINE MODEL OF GENITAL INFECTION

Introduction

Chlamydia trachomatis is the causative agent of the most commonly reported

sexually transmitted disease in the United States, Chlamydia, and a public health threat

worldwide [56] [63] [147] [222]. Despite the overwhelming global presence of

chlamydial infection, many cases are undiagnosed due to asymptomatic infection and

may serve as a reservoir for infection [175] [223]. Regardless of whether Chlamydia

trachomatis infection is symptomatic or asymptomatic, women with genital infections

may develop long-term sequelae like pelvic inflammatory disease (PID), ectopic

pregnancy, involuntary infertility, and miscarriage [169] [170]. Antibiotic therapy is

currently effective in clearing the organism from the lower genital tract but the

emergence of antibiotic resistance, although rare, is a viable concern [224] [225] [226]

227]. Currently, rapid antibiotic intervention is a key component of Chlamydia control

programs [173] [175] and may serve as the selective pressure necessary for the evolution

of antibiotic resistant human strains. Taken together, the high rate of asymptomatic

infection and the potential for developing antibiotic resistance despite the current

availability of effective antimicrobial therapies underscore the need for a safe and

efficacious vaccine.

Trachoma vaccine trials using whole organisms, both inactivated and live, were

performed decades ago and are perhaps the most notable vaccination studies to date.

Unfortunately, a subset of individuals who were vaccinated and reexposed to Chlamydia

experienced significantly more severe disease when compared to non-vaccinated cohorts

[124] [228] [229]. While no human trials have been untaken since the trachoma study,

researchers have continued the quest to identify safe chlamydial antigens that elicit

immunological memory- the basis of all vaccines.

A number of Chlamydia trachomatis- associated antigenic proteins have been

described, particularly proteins associated with the cell surface. Many studies have

focused on developing a subunit-based vaccine using Chlamydia trachomatis major outer

membrane protein (MOMP) as the antigen [230] [231]. MOMP has been an attractive

candidate because it constitutes an approximated 60% of the total outer membrane mass

and is immune-assessable due to its location on the chlamydial surface [21] [125].

Although MOMP seemed promising, its ability to induce protection has been sketchy at

best, displaying varying protection in small animal models and higher order primates

[232] [233] [234] [235] [236] [237] [238] [239] [240] [241]. There may be several

reasons for the variation seen in the previously mentioned studies including but not

limited to the animal model and strain(s) used, immunization site, challenge route,

definition of protection (I.E. reduction in shedding, fertility, cytokine production,

antibody titer, or sequelae formation) , and antigen preparation. Indeed, one of the main

lessons taken from previous MOMP studies is that conformational integrity of the

chlamydial membrane may increase protection following chlamydiae challenge. As a

51

result, we elected to use detergent-extracted chlamydial outer membrane complexes

(COMCs) as described by Caldwell et al [125] in the preliminary studies outlined later in

this Chapter. Sarkosyl-Insoluble COMC was found to be structurally intact and

maintained the shape observed in protein-rich infectious chlamydial particles, elementary

bodies (EBs).

In the pages to follow, I outline experiments evaluating the efficacy of COMC as

a potential antigen in both systemic and genital models of chlamydiae infection. As a

disclaimer, these studies are in their infancy at best and should not be interpreted as

complete studies. Several gaps exist in the experiments outlined hereafter but these data

do provide interesting tangents that may serve as the basis for future experimental

direction. To that end, our initial results in the systemic model showed promise by

negating the effects of lethal interperitoneal chlamydial doses. Conclusions originating

from studies in the urogenital murine model were more muddled when compared to the

lethal model. Despite the inconclusive nature of the genital tract model results, we

examined the potential benefits of alum, an adjuvant known to generate a TH2 cell-

mediated response, in the prevention of severe genital sequelae.

Materials and Methods (Systemic Lethal Model)

Mice

Female DBA/2J mice were purchased from Jackson Laboratories at 7-8 weeks of

age. They were housed in the University of Tennessee Health Science Center animal

facility and animal protocols were performed in compliance with institutional IACUC

and federal mandates.

Chlamydiae

Chlamydia psittaci 6BC was propagated in the murine fibroblast cell line L929

(ATCC; Manassas, VA) and stored at -80o C. Titers were determined by infecting L929

cells plated at a density of 2 x 105 cells/well in 24- well plates (Corning Costar

Corporation, Cambridge, MA), with tenfold dilutions of thawed stock. Infected

monolayers were incubated for 48 hours, methanol fixed, and chlamydial inclusions were

enumerated using fluorescent staining with FITC-conjugated anti-chlamydial

lipopolysaccride antibody (Fitzgerald Industries Internation Incorporated, Concord, MA).

COMC Extraction

Chlamydia psittaci 6BC COMC was purified as described by Caldwell et al [125].

In brief, C. psittaci 6BC stock was thawed at room temperature and suspended in 5mL of

pH 8 phosphate buffered saline (PBS) containing 2% dipolar ionic Zwittergonic detergent

and 1.5 mm EDTA. A dry ice bath was used to precipitate proteins and the resulting

52

suspension was sonicated, in series, 3 times with 1 minute iced incubation periods

between each round of sonication. The cell suspension was incubated for 3 hours at 37o

C and ultracentrifuged at 32, 700 rotations per minute (RPM) for 1hour.

Immunization and Infection

DBA/2J mice were intramuscularly mock immunized with 100mL of sterile

Dulbecco’s phosphate buffered saline (PBS), viable whole elementary bodies (EBs) of

6BC (5 x 102 IFU in 100mL of sterile PBS), or 6BC-derived COMC (40mg total) in

100mL of PBS, respectively. Mice received a total of two immunizations at days -14 and

-7. On day 0, mice were intraperitoneally challenged with a low dose (1 x 103 IFU,

equivalent to 1000 LD50) or a high dose (1 x 105 IFU, equivalent to 100000 LD50) and

monitored for survival. Surviving animals were sacrificed 15 days post infection

irrespective of immunization regimen.

Results (Systemic Lethal Model)

Our laboratory was instrumental in developing a systemic interperitoneal model

of chlamydial disease using C. psittaci 6BC, a zoonotic to which mice are highly

susceptible. Using the systemic model Byrne and colleagues were able to elucidate basic

concepts concerning interferon-gamma and Chlamydia psittaci clearance [242] [243]

[244]. Unpublished data from our laboratory showed that doses as low as 1 EB can cause

mice to succumb to infection within 10 days of initial C. psittaci challenge. These data

provided the basis for using the systemic model of chlamydiae infection to elucidate

COMC immunogenicity. One hundred percent of mice immunized with viable 6BC

Chlamydia psittaci elementary bodies (EBs) survived challenge and recovered from

infection irrespective of the challenge dose. Conversely, all mock PBS immunized mice

succumbed to infection within 11 days of interperitoneal challenge. 20% of COMC

antigen immunized mice survived an inordinately high dose challenge and went on to

recover from infection (Figure 4-1). Moreover, 60% of mice immunized with 6BC

Chlamydia psittaci -derived antigen survived and recovered from lethal challenge

(Figure 4-2).

Despite the rigor associated with the systemic chlamydial intraperitoneal murine

model, COMC preparations were capable of eliciting partial protection against lethal

challenge. Both immunizing doses, 102 and 105 IFU, supersede the LD50 and highlight the

sensitivity of DBA/2J inbred mice to 6BC Chlamydia psittaci intraperitoneal infection as

previously illustrated by the Byrne laboratory. Collectively, these data suggested COMC-

induced partial protection with homologous challenge and justified transitioning these

studies into the urogenital model which would display more subtle read-outs.

53

Figure 4-1. High dose 6BC C. psittaci systemic infection survival curve.

High Dose 6BC C. psittaci Interperitoneal Challenge

0 3 6 9 12 150

20

40

60

80

100

120

LIVE 6BC EB

Antigen

PBS

Time:Days

Perc

en

t su

rviv

al

54

Figure 4-2. Low dose 6BC C. psittaci systemic infection survival curve.

Low Dose 6BC C. psittaci Interperitoneal Challenge

0 3 6 9 12 150

20

40

60

80

100

120

LIVE 6BC EB

Antigen

PBS

Time: Days

Percen

t su

rviv

al

55

Materials and Methods (Urogenital Model)

Mice

Female DBA/2J mice were purchased from Jackson Laboratories at 7-8 weeks of

age. They were housed in the University of Tennessee Health Science Center animal

facility and animal protocols were performed in compliance with institutional IACUC

and federal mandates.

Chlamydiae

Chlamydia muridarum (Weiss) was propagated in the human epithelial cell line

HeLa 229 (ATCC; Manassas, VA) and stored at -80o C. Titers were determined by

infecting HeLa 229 cells seeded at a density of 2 x 105 cells/well in 24- well plates

(Corning Costar Corporation, Cambridge, MA), with tenfold dilutions of thawed stock.

Infected monolayers were incubated for 48 hours, methanol fixed, and chlamydial

inclusions were enumerated using fluorescent staining with FITC-conjugated anti-

chlamydial lipopolysaccride antibody (Fitzgerald Industries Internation Incorporated,

Concord, MA).

COMC Extraction

Chlamydia muridarum COMC was purified as described by Caldwell et al [125].

In brief, C. muridarum stock was thawed at room temperature and suspended in 5mL of

pH 8 phosphate buffered saline (PBS) containing 2% dipolar ionic Zwittergonic detergent

and 1.5 mm EDTA. A dry ice bath was used to precipitate proteins and the resulting

suspension was sonicated, in series, 3 times with 1 minute iced incubation periods

between each round of sonication. The cell suspension was incubated for 3 hours at 37o

C and ultracentrifuged at 32, 700 rotations per minute (RPM) for 1hour.

Immunization and Infection

DBA/2J mice were intramuscularly immunized with 100mL of sterile Dulbecco’s

phosphate buffered saline (PBS), 100mL of sterile Dulbecco’s phosphate buffered saline

(PBS) and 25ug of alum, live C. muridarum elementary bodies (5 x 102 IFU in 100mL of

sterile PBS), 25ug of Chlamydia muridarum-derived COMC in 100mL of PBS, or 25ug

of Chlamydia muridarum-derived COMC in 100mL of PBS and 25ug of alum. Mice

received a total of two immunizations at days -14 and -7. One week prior to challenge,

the estrous cycle was synchronized by subcutaneous injection of 2.5mg of

medroxyprogesterone acetate (Sicor, CA). On day 0, mice were intravaginally challenged

with 2 x 103 IFU, a non-lethal dose of C. muridarum.

56

Results (Urogenital Model)

C. muridarum and C. psittaci-derived COMC preparations were compared by

commassie-stained SDS-Page gel and displayed comparable banding patterns

(Figure 4-3). As expected, animals intramuscularly immunized with viable whole C.

muridarum elementary bodies and intravaginally challenged with 5 x 103 IFU displayed

the most severe disease when compared to other immunization groups as evidenced by

dilation of the uterine horns and reddening of the genital tract (Figure 4-4A). Mock

immunized animals (Figure 4-4B) also displayed fluid accumulation in the uterine horn,

although to a lesser extent when compared to the live immunized group. COMC

immunized animals displayed less severe disease when compared to live immunized

groups but still displayed mild to moderate dilation and reddening (Figure 4-4D). While

animals immunized with COMC-alum displayed mild reddening, dilation was not

observed (Figure 4-4E). Lastly but most intriguingly, animals immunized with the PBS-

alum combination displayed no dilation or substantial reddening (Figure 4-4C).

While we hypothesized that alum would augment protection elicited by COMC

antigen immunization despite its polarity toward a Th2-type response, we were perplexed

by the observation seen in PBS-alum immunized groups. Unexpectedly, the alum

adjuvant seemed to negate the development of severe genital tract sequelae induced by

exposure to C. muridarum intravaginal infection. The study described below followed as

an attempt to assess whether alum alone was capable of negating the development of

upper genital tract sequelae following C. muridarum intravaginal infection.

Materials and Methods (Alum Study)

Mice

Female C57BL/6J mice were purchased from Jackson Laboratories at 7-8 weeks

of age. They were housed in the University of Tennessee Health Science Center animal

facility and animal protocols were performed in compliance with institutional IACUC

and federal mandates.

Chlamydiae

Chlamydia muridarum (Weiss) was propagated in the human epithelial cell line

HeLa 229 (ATCC; Manassas, VA) and stored at -80o C. Titers were determined by

infecting HeLa 229 cells seeded at a density of 2 x 105 cells/well in 24- well plates

(Corning Costar Corporation, Cambridge, MA), with tenfold dilutions of thawed stock.

Infected monolayers were incubated for 48 hours, methanol fixed, and chlamydial

inclusions were enumerated using fluorescent staining with FITC-conjugated anti-

chlamydial lipopolysaccride antibody (Fitzgerald Industries Internation Incorporated,

Concord, MA).

57

Figure 4-3. Comassie stained SDS-Page gel of C. muridarum (MoPn)-derived

COMC and C. psittaci (6BC)-derived COMC preparations.

Note: Chlamydiae COMC preparations were extracted as previously described in Chapter

4. Protein concentrations were determined by Modified Lowry Protein Assay and

visualized by SDS-Page gel. Molecular weight protein standard (shown on left) were

used to approximate the molecular weight of visualized bands.

MoPn 6BC

250 kD

150 kD

100 kD

75 kD

50 kD

37 kD

25 kD 20 kD

15 kD

10 kD

58

Figure 4-4. C. muridarum-derived COMC immunization study gross pathology.

Note: Representative photos of genital gross pathology post homologous C. muridarum immunization and challenge. Photo (A) is

representative of live immunizations. Photo (B) is representative of mock immunized animals. Photo (C) is representative of mock-

alum immunized animals. Photo (D) is representative of COMC immunized animals and photo (E) represents animal cohorts

immunized with COMC-alum. Black arrows point out selected areas of dilatation, reddening, and hydros0alpinx formation.

59

Immunization and Infection

C57BL/6J mice were intramuscularly immunized with 100mL of sterile

Dulbecco’s phosphate buffered saline, 100mL of sterile Dulbecco’s phosphate buffered

saline and 25ug of alum (Thermo Scientific), live C. muridarum elementary bodies (5 x

102 IFU in 100mL of sterile PBS), or a mixture of live C. muridarum (5 x 102 IFU in

100mL of sterile PBS) and 25ug of alum. Mice received two immunizations one week

apart and were treated subcutaneously with 2.5mg of medroxyprogesterone acetate

(Sicor, CA) at days -9 and -3 to synchronize the esterous cycle and enhance mouse

susceptibility to chlamydial infection. On day 0, mice were intravaginally challenged

with 2 x 103 IFU Chlamydia muridarum in 10uL of PBS. Animals that were immunized

and challenged were sacrificed 35 days post infection. Immunized animals that did not

receive an infection were sacrificed 21 days after day 0.

Pathology Assessment

Before removing the genital tissue, an in situ gross examination was performed

for evidence of chlamydial-induced abnormalities such as uterine horn dilation and

hydrosalpinx formation. Genital tracts were aseptically removed, photographed,

subjected to macroscopic inspection, and assigned a severity score on the day of sacrifice.

Results (Alum Study)

Immunogens without Intravaginal Chlamydia muridarum Challenge Do Not Induce

Severe Upper Genital Tract Sequelae Formation

Female C57BL/6J mice were intramuscularly immunized with alum adjuvant,

viable C. muridarum and alum adjuvant, viable C. muridarum, or mock immunized with

sterile PBS according to the timeline displayed in (Figure 4-5). Genital tracts from each

animal were aseptically removed, weighed, photographed, and grossly assessed for upper

genital tract sequelae formation three weeks after day 0. Two non-immunized C57BL/6

mice, of the same age and gender, were used as a reference. As hypothesized,

hydrosalpinx formation was not observed in any of the immunization groups (Figure

4-6). Negligible increases in dilation of the uterine horns were observed in mock, C.

muridarum and alum adjuvant, and C. muridarum alone immunized animals. Although

these increases were minimal, they were documented by granting the lowest severity

score possible (Figure 4-7). Overall, genital tract tissues, regardless of immunization

group were comparable to that observed in the two non-immunized, non-challenged

reference samples.

60

Figure 4-5. Alum study experimental timeline.

61

Figure 4-6. Oviduct pathology severity of non-challenged immunized groups.

Note: Solid line represents the mean of each immunogen group. Symbols represent

individual mice (N=5 per group except for normal control).

62

Figure 4-7. Uterine horn pathology severity of immunized, non-challenged

groups.

Note: Solid bars represent the mean severity score for each immunogen group. Symbols

represent individual animals (N=5 per group except for normal controls).

63

Alum Alone Does Not Negate the Development of Severe Upper Genital Tract

Complications

Female C57BL/6J mice were intramuscularly immunized with alum adjuvant,

viable C. muridarum and alum adjuvant, viable viable C. muridarum, or mock

immunized with sterile PBS according to the timeline displayed in (Figure 4-5). All

immunization groups were intravaginally challenged with 2 x 103 IFU of Chlamydia

muridarum. Genital tracts from each animal were aseptically removed, weighed,

photographed, and grossly assessed for upper genital tract sequelae formation and

severity 35 days post infection. Three female C57BL/6 mice which were not immunized

or challenged were used as a negative reference.

Unlike their non-challenged counterparts (Figure 4-6), all immunization groups

displayed some level of oviduct disease (Figure 4-8). 40% of mock immunized animals

displayed unilateral hydrosalphinx formation while 20% displayed bilateral oviduct

involvement. 20% of animals immunized with C. muridarum or C. muridarum and alum

adjuvant exhibited unilateral oviduct disease. Interestingly, 20% of animals immunized

with alum adjuvant alone displayed bilateral hydrosalpinx while unilateral oviduct

pathology was observed in 40% of the same immunization group.

Uterine horn dilation was seen in all groups immunized (Figure 4-9). 60% of

animals immunized with C. muridarum alone displayed mild dilation with a mean

severity score of approximately 1. Eighty percent of animals immunized with C.

muridarum and alum adjuvant were found to have dilated uterine horns with 60% of

those being mildly dilated and 20% displaying moderate dilation. Only 20% of alum

adjuvant immunized animals displayed uterine involvement, whereas, 60% of mock

immunized animals experienced uterine horn dilation.

Discussion

Although C. trachomatis lower genital tract infections may resolve without

adverse reproductive complications [164] [245] [246], a subset of those infected may

develop upper genital tract sequelae. Presently, Chlamydia control initiatives focus on

identifying and treating individuals before, at least in humans, irreversible tissue damage

occurs. This approach is complicated by the fact that most human chlamydial infections

are often asymptomatic. As a result, the most effective way of controlling chlamydiae

infections would be to develop a vaccine. Although the studies outlined in this Chapter

fail to provide definitive answers pertaining to protection against chlamydial infection,

they do highlight the need for ongoing vaccine development initiatives to consider the

pathogenesis approach given the overarching goal of any Chlamydia trachomatis

vaccination effort would be to preserve reproductive health by preventing the

development of severe disease manifestations.

In the systemic model of chlamydial infection we showed that a detergent-

extracted preparation of C. psittaci-derived COMC protected mice from lethal

intraperitoneal challenge. To our knowledge, this is the first study of its kind and the

64

Figure 4-8. Oviduct pathology severity of immunized and challenged groups.

Note: Solid bars represent the mean severity score for each immunogen group. Symbols

represent individual animals (N= 5 per group except for normal control).

Norm

al

PBS

Alu

m

Alu

m/M

oPn

MoP

n

-1

0

1

2

3

4

5

Immunogen

Severi

ty S

co

re

65

Figure 4-9. Uterine horn pathology severity of immunized and challenged groups.

Note: Solid bar represents the mean for each immunogen group. Symbols represent

individual animals (N = 5 per group except for normal control).

Untr

eate

d

PBS

Alu

m

Alu

m/M

oPn

MoPn

-1

0

1

2

3

4

5

Immunogen

Severi

ty S

co

re

66

results inspired us to examine the efficacy of COMC in the C. muridarum urogenital

model given the fact that mice intravaginally infected with Chlamydia muridarum

develop genital sequelae similar to that observed in human populations. Seeing as the

development of genital morbidities such as hydrosalpinx and endometritis are more

subtle than the mortality (survival vs death) end point observed in the systemic model, we

opted to co-administer alum adjuvant in subsequent genital tract experiments as a way of

enhancing the responses observed with homologous COMC antigen. Additionally,

aluminum hydroxide, aluminum phosphate, and aluminum sulfate are the only vaccine

adjuvant formations licensed in the United States and are currently being used in vaccines

directed against the intracellular pathogens, hepatitis B virus (HBV) and human

papilloma virus (HPV) [247].

In our studies, COMC-alum immunized groups (Figure 4-1E), as hypothesized,

fared better than mock immunized groups (Figure 4-1B) and live immunized groups

(Figure 4-1A) displaying no increases in uterine horn or hydrosalpinx formation. As we

hypothesized, DBA/2J mice immunized with live C. muridarum and challenged with C.

muridarum displayed the most severe pathology with both endometrial dilation and

oviduct inflammation (Figure 4-1A). These findings complement our studies and the

studies of others that suggest chlamydial infection recurrence increases the risk for

reproductive sequelae [177] [179] [248].

By far the most interesting observation was the fact that alum adjuvant and PBS

immunized animals displayed no signs of chlamydial-induced pathology or reddening

which is indicative of inflammation. While Th2-driven responses such as those prompted

by the alum adjuvant have been shown to promote immunopathology in chronic diseases

such as schistosomiasis [249] [250], autoimmune diseases, and allergies, more recent

studies have implicated TH2-linked responses to tissue repair and wound healing. As a

result, we hypothesized that a Th2-type response may play a protective role in upper

genital tract disease formation.

To experimentally address the effects of alum adjuvant on chlamydial-induced

genital tract pathology we immunized female C57BL/6J mice with alum adjuvant, alum

and whole organism C. muridarum, or intact C. muridarum organisms alone. Based on

the observations seen in the COMC urogenital studies, we hypothesized that alum only

severity scores would be on par with severity scores associated with non-immunized,

non-challenges animals (negative controls). Interestingly, animals immunized with alum

adjuvant alone displayed severity scores comparable to those observed in alum/C.

muridarum and C. muridarum alone immunization groups (Figure 4-9). While the alum

alone observation was unexpected, we were encouraged by the fact that all immunization

groups exhibited less severe disease, as indicated by mean severity score, than the mock-

immunized PBS group which complemented the results seen in the preliminary COMC

urogenital studies (Figure 4-4 and Figure 4-9).

There are at least two, not mutually exclusive, explanations for the discrepant

results seen in the alum adjuvant alone group. One is the mouse inbred strain we used in

the follow-up studies. We substituted the DBA/2J mouse strain originally used in the

67

COMC studies for the C57BL/6J inbred strain because B6 animals used in ongoing,

unrelated studies in our laboratory routinely exhibit more severe genital tract disease

when compared to DBA/2J animals. We hypothesized that by using the ‘more severe’

B6 background we would stratify the results seen between groups, making the potential

benefits of alum immunization easier to identify. We know that the enhancement effects

of alum are less pronounced than that of other adjuvants like Freud’s complete adjuvant

(FCA) and are optimal when used in conjunction with an immunogenic compound.

Assuming the upper genital tract sequelae observed in our model is mediated, at least in

part, by the host immune response, it is possible that the B6 TH1-bias was too much for

alum adjuvant to overcome which manifested as mild disease. Secondly, but not less

important, is the chlamydial preparation used in the study. Chlamydiae-derived outer

membrane complex preparations were used as immunogen in the preliminary studies

while whole viable organisms were used in the alum efficacy studies. This is important in

that the alum alone immunized group scores (Figure 4-9) are indistinguishable from

Chlamydia muridarum-alum and Chlamydia muridarum alone group scores. In using

concentrated COMC preparations, we directed the host response against known

immunogenic proteins associated with the chlamydial membrane. Perhaps statistical

differences were not observed amongst immunization groups, including alum alone,

because we diversified the anti-chlamydial antibody pool by using whole organisms over

purified COMC preparations. While it is conceivable that either or both of these

explanations contributed to differences seen in our alum efficacy experiments, further

study is needed to draw definitive conclusions.

While data presented in this Chapter would, seemingly, negate the hypothesis put

forth in Chapter 2 relating to the potential benefits of having TH2-related IL-4 present late

during recurrent infection that is not the case. Our hypothesis hinges on the idea that IL-4

is detectable late during recurrent infection cycles as a way for chlamydial hosts to repair

severe tissue damage. In our studies, alum adjuvant was administered concurrently with

chlamydial immunogen, prior to the development of severe upper genital tract

complications. It is logically to hypothesize that a portion of time in which the host

perceives ‘self-damage’ is necessary for the gearing up of potentially protective TH2-

related responses. As stated previously, this is only a hypothesis albeit formed from data

derived from ongoing chlamydial genital studies in our laboratory. The continuation and

translational relevance of the studies reported in this Chapter are currently being

investigated in our laboratory and are supported, at least in part, by the recent report

published by Rodolfo et al suggesting an evolutionary genital tract bias for Th2 responses

as a mechanism to prevent chlamydial-induced genital tract complications in humans

[195].

68

CHAPTER 5. CONCLUSIONS

The reproductive health of women is of particular concern with current CDC

Chlamydia surveillance data indicating increases in reported chlamydial infections,

perhaps, in part, as a result of rapid antibiotic intervention. To this end, we interrogated

the murine model for its potential to serve as a translational bridge between population-

based epidemiology and previous animal and human studies. Our studies reaffirm the

benefit of rapid antibiotic intervention in Chlamydia control efforts and suggest an added

benefit during repeated infection even in the absence of antibiotic treatment. Moreover,

our results suggest a TH2-independent role for IL-4 in genital tract tissue repair.

Testing the IL-4 Hypothesis

IL-4 as a mediator of TH2-driven immunity is well established but the role IL-4

may play in genital tract tissue repair is largely unknown. In our studies we show

elevated levels of systemic IL-4 late during recurrent chlamydial genital infection and

cellular infiltrates that are known to produce IL-4 in the genital tract. We also show a

correlation between systemic IL-4 and disease severity. To assess the role of IL-4 in the

arrested immunity model, longitudinal studies are necessary. In these studies we would

measure the onset, duration, and resolution of TH1 and TH2-linked cytokines and disease

progression. This is important given the fact that tissue repair mechanisms rely on the

presence of tissue damage. If IL-4 is indeed a driver of in vivo tissue repair, at least in

this model, it is plausible that the production and secretion of the cytokine would begin

with or after the development of chlamydial-induced pathology. Future studies should

also measure IL-4 transcription levels in implicated cell types (mast cells and

eosinophils) to confirm that they are the primary source of IL-4. These studies would be

followed by IL-4 knockout studies that examine the effect of IL-4 on collateral tissue

damage during multiple rounds of chlamydiae infection.

Identifying Genetic Link to Age-Dependent Disease Severity

Studies are currently underway using B (C57BL/6) x D (DBA/2J) recombinant

inbred mouse strains to identify genetic factors that may modulate disease susceptibility

in an age-dependent manner. By interrogating the GeneNetwork database using a forward

genomic approach, we hope to identify genes associated with the modulation of oviduct

and uterine horn disease severity in an age and strain dependent manner. If disease

severity is associated with specific genetic loci, human cell culture studies focused on

RNA silencing will ensue. These studies will be followed with in vivo studies using

knockout mice specific for the identified gene(s).

69

Implications for Vaccine Development

It is generally accepted that an effective anti-chlamydial vaccine would need to

elicit a robust TH1-type CD4+ T cell response but our studies suggest that prophylactic

control of chlamydiae may be more complex than originally appreciated. If the goal is to

clear the pathogen from the site of infection and minimize the likelihood of developing

adverse reproductive upper genital tract complications, eliciting TH1-linked immune

responses may not be enough. It may be beneficial for the host to mount a mixed

immunological response (I.E. TH1/ TH2). Our work highlights the need for additional

studies aimed at addressing the role of mixed cell-mediated responses in chlamydial

infection and immunopathological genital tract damage.

70

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91

APPENDIX A. MORBIDITY AND PATHOLOGY SEVERITY POST-

PRIMARY INFECTION IN DEFERRED TREATMENT STUDIES

Note: C57BL/6 female mice were purchased from Jackson Laboratories at 7-8 weeks of

age. Animals were subcutaneously treated with progesterone at days 14 and 7 per

intravaginal challenge. On day 0, animals were intravaginally challenged with 2 x 103

IFU C. muridarum. UN refers to animals that did not receive treatment during the

courseo f the experiment. (0-14) represents animals that were intraperitoneally treated

with doxycycline starting at day 0 for 14 days consecutively. (2-16) represents animals

that were intraperitoneally treated with doxycycline starting 2 days post primary

challenge for 14 days consecutively. (4-18) represents animals that were intraperitoneally

treated with doxycycline starting on day 4 post primary challenge for 14 days

consecutively. Normal represents negative the negative control group which neither

received treatment nor intravaginal Chlamydia muridarum challenge. Animals were

sacrificed 56 post-primary infection.

92

APPENDIX B. MORBIDITY AND PATHOLOGY SEVERITY POST-

SECONDARY INFECTION IN DEFERRED TREATMENT STUDIES

Note: C57BL/6 female mice were purchased from Jackson Laboratories at 7-8 weeks of

age. Animals were subcutaneously treated with progesterone at days 14 and 7 per

intravaginal challenge. On day 0, animals were intravaginally challenged with 2 x 103

IFU C. muridarum. UN refers to animals that did not receive treatment during the course

of the experiment. (0-14) represents animals that were intraperitoneally treated with

doxycycline starting at day 0 for 14 days consecutively. (2-16) represents animals that

were intraperitoneally treated with doxycycline starting 2 days post primary challenge for

14 days consecutively. (4-18) represents animals that were intraperitoneally treated with

doxycycline starting on day 4 post primary challenge for 14 days consecutively. Normal

represents negative the negative control group which neither received treatment nor

intravaginal Chlamydia muridarum challenge. Animals were sacrificed 56 post-

secondary infection.

93

APPENDIX C. TH1-RELATED CYOKINES POST RECURRENT

INFECTION IN ARRESTED IMMUNITY MODEL

94

APPENDIX D. TH2-RELATED CYTOKINES POST RECURRENT

INFECTION IN ARRESTED IMMUNITY MODEL

95

VITA

Enitra N. Jones was born in Houston, Texas in 1983, the daughter of Ezra C.

Jones and Evelyn T. Jones. In 2001, she graduated from West Jefferson High School of

Harvey, Louisiana in the top three percent of her class. In July 2005, Enitra earned her

Bachelor of Science degree in Biology with a concentration in Microbiology from the

Honors College at Southern University and Agricultural and Mechanical College in

Baton Rouge, Louisiana (Cum Laude Latin Honors). In 2006, Enitra enrolled in the

Integrated Program of Biomedical Sciences at the University of Tennessee Health

Science Center in Memphis, Tennessee. She graduated in 2013 with a Doctorate of

Philosophy in Microbial Pathogenesis, Immunology, and Inflammation. Enitra is

currently a two-year American Public Health Laboratories (APHL)/ Centers for Disease

Control and Prevention (CDC) Emerging Infectious Diseases (EID) Postdoctoral

Research Fellow in Atlanta, Georgia.