Identification of Cytokine Profiles Associated with Endometrial Chlamydia Infection

IDENTIFICATION OF CYTOKINE PROFILES ASSOCIATED WITH ENDOMETRIAL CHLAMYDIA INFECTION

De’Ashia Elizabeth Lee

A thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department of Microbiology and Immunology in the School of Medicine.

Chapel Hill 2018

Approved by:

Toni Darville

Nilu Goonetilleke

Jason Whitmire

Barbara Salvodo

ABSTRACT

De’Ashia Elizabeth Lee: Identification of Cytokine Profiles Associated with Endometrial Chlamydia Infection (Under the direction of Toni Darville and Nilu Goonetilleke)

Chlamydia trachomatis (CT) infection can lead to reproductive tract morbidities when it ascends to the upper genital tract of women, and repeated infections worsen disease. Cervical cytokines associated with disease or infection susceptibility in women are unknown. Forty-eight cytokines were measured in cervical secretions of 160 women with CT infection, 68 who had endometrial infection, and 92 with cervical infection only. Participants were monitored for repeat

CT infections over the following year. Multivariable stepwise regression examined whether cytokines were associated with endometrial infection at the enrollment visit or reinfection. IL-16, was associated with decreased risk of endometrial infection while CXCL10, CXCL13, and

TNFα, were associated with increased risk of endometrial infection. Although we did not identify cytokines significantly associated with an altered risk of repeat infection, VEGF, a T cell chemokine, and IL-14, a B cell chemokine, were associated with decreased and increased risk of reinfection by univariable analysis, respectively.

iii

ACKNOWLEDGEMENTS

To my family and friends, thank you for all the unconditional love and support throughout my life. Your unwavering, selfless support offers me endless motivation to overcome any obstacle. In your own unique ways, each one of you has taught me that to an unstoppable force, there is no immovable object.

To my mentors, past and present, thank you for your guidance and mentorship. I appreciate the dedication and support that you all have offered me throughout the years. To Toni and Nilu, thank you for the opportunity to become a member of your labs. I have learned so much from the both of you, and I am sure these lessons will serve me well in the future. To Jason and Barbara, thank you for the generosity, feedback, and willingness to serve on my committee.

To Ashalla, Kim, and Jessica, thank you for listening, understanding, motivating, and supporting me throughout the years. This would not be possible without you! To Bob, thank you for your dedication to the students, I appreciate all that you have done and continue to do.

I would like to thank all the women who participated in TRAC. Without your involvement and cooperation, I would not have been able to conduct this analysis. Additionally, I would also like to thank the members of the Immunology unit of the Duke Regional

Biocontainment Laboratory for their assistance in running all the panels. Finally, I would like to thank the biostatisticians on this project for their excellent guidance and support during this process. Jing, Wujuan, and Li, your assistance has been invaluable. I could not have done this without you!

TABLE OF CONTENTS

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

LIST OF ABBREVIATIONS ...... ix

Chapter 1: History ...... 1

Lifecycle ...... 2

Serovar classifications ...... 2

Clinical manifestations ...... 3

Diagnosis ...... 3

Epidemiology ...... 4

Pelvic Inflammatory Disease ...... 4

Current treatment options ...... 6

Innate immunity ...... 6

Toll-like receptors ...... 6 Chlamydial plasmid...... 7 Matrix metalloproteinases ...... 7 Neutrophils ...... 8 Cytokines and Chemokines ...... 9 IL-1 ...... 9 TNFα ...... 10 IL-17A ...... 11 Chemokines ...... 12 Adaptive immunity ...... 13

Humoral immunity ...... 13 IgG in human studies ...... 14

v IgA in mouse models ...... 14 IgA in human studies ...... 15 Cell-mediated immunity ...... 15 CD4+ T cells ...... 15 CD4+ T cells and protection in mice ...... 15 CD4+ T cells and protection in humans ...... 16 CD4+ T cells and pathology in mice ...... 17 CD4+ T cells and pathology in humans ...... 18 CD8+ T cells ...... 18 CD8+ T cells and protection in mice ...... 18 CD8+ T cells and protection in humans ...... 19 CD8+ T cells and pathology in mice ...... 20 CD8+ T cells and pathology in humans ...... 21 Summary of immune responses associated with chlamydia-induced pathology ...... 22 Current study ...... 24

INTRODUCTION ...... 24

METHODS ...... 27

Study population ...... 27 Definition of clinical and microbiological subgroups ...... 28 Quantification of cytokines in cervical secretions ...... 29 Statistical Analysis ...... 31 Preliminary statistical analysis ...... 31 Relationship between cervical cytokines and endometrial infection...... 32 Relationship between cervical cytokines and reinfection...... 33 RESULTS ...... 34

Baseline characteristics of participants ...... 34 Association between cervical cytokines and endometrial infection ...... 34 Association between cervical cytokines and reinfection ...... 35 DISCUSSION ...... 36

FUTURE DIRECTIONS ...... 40

REFERENCES ...... 59

vi LIST OF TABLES

Table 1: Univariable regression analysis: Association of cytokines with endometrial chlamydia infection in women with high bacterial burden ...... 45

Table 2: Sociodemographic characteristics of cohort ...... 47

Table 3: Descriptive statistics of cytokine levels ...... 49

Table 4: Association of cytokines with chlamydia burden by univariable regression analysis ...... 51

Table 5: Association of cytokines with chlamydia endometrial infection by univariable regression analysis ...... 53

Table 6: Cytokines associated with chlamydia endometrial infection by multivariable stepwise regression ...... 55

Table 7. Association of cytokines with chlamydia reinfection by univariable regression analysis ...... 56

vii LIST OF FIGURES

Figure 1: Proposed model of endometrial Chlamydia infection ...... 58

viii LIST OF ABBREVIATIONS

APC Antigen presenting cell

BV Bacterial vaginosis

CDC Center for Disease Control and Prevention

EB Elementary Body

ECM Extracellular matrix

HLA Human leukocyte antigen

HR Hazard ratio

Hsp60 Heat shock protein 60

Ig Immunoglobulins i.p. Intraperitoneal i.v. Intravenous

LPS Lipopolysaccharide

MHC Major histocompatibility complex

MIF Microimmunofluorescence

MMP Matrix metalloproteinase

MOMP Major outermembrane protein

MSM Men who have sex with men

NAAT Nucleic Acid Amplification Test

ix OR Odds ratio

PAMP Pathogen Associated Molecular Pattern

PBMC Peripheral blood mononuclear cell

PID Pelvic Inflammatory Disease

PRR Pathogen Recognition Receptor

RB Reticulate Body

SCID Severe combined immunodeficiency

SNP Single Nucleotide Polymorphism

STI Sexually Transmitted Infection

TCR T cell receptor

TFI Tubal Factor Infertility

TLR Toll-like Receptor

TRAC T cell Response Against Chlamydia

Tfh T follicular helper cell

Treg Regulatory T cell

WHO World Health Organization

Chapter 1: History

Chlamydia trachomatis is an obligate intracellular, Gram-negative bacterium that causes a wide range of diseases in humans. It was first discovered in 1907 by Ludwig Halberstaedter and Stanislaus von Prowazek, who observed intracytoplasmic inclusions in conjunctival scrapings from subjects with trachoma. The scrapings were used to infect the eyes of orangutans, demonstrating that disease could be introduced into a healthy animal. Due to its intracellular nature, C. trachomatis was initially thought to be a protozoan and then a virus. It was not until the 1960s that it was accurately acknowledged as a bacterium. C. trachomatis was first isolated from trachoma patients in 1957 by T’ang and colleagues by cultivating the bacterium in the yolk sac of embryonated chicken eggs 1. Soon after, Jones and colleagues isolated the organism from patients with ocular and genital disease by also propagating the bacterium in hen’s eggs2. By

1965, C. trachomatis propagation in cell culture was achieved by Gordan and Quan 3. Gordan and Quan isolated chlamydia using irradiated McCoy cells, a fibroblast cell line, and centrifugation. The cell culture method was instrumental in defining C. trachomatis serovars that are distinguished by variant regions in the major outer membrane protein (MOMP) (see “Serovar classifications”) and enabled the confirmation of C. trachomatis as a cause of non-gonococcal urethritis in the United Kingdom and the United States in the 1970s. Despite this success, cell culture was very time consuming (see “Lifecycle”) and relatively insensitive. The introduction of nucleic acid amplification tests (NAATs) in the mid-1990s, dramatically improved the diagnostic sensitivity of C. trachomatis.

1 Lifecycle

Chlamydia trachomatis has two morphologically and functionally distinct forms, the extracellular infectious elementary body (EB) and the intracellular non-infectious reticulate body

(RB). The life cycle of C. trachomatis is approximately 48 - 72 hours and starts when an EB attaches to a host epithelial cell. The EB is endocytosed into a membrane-bound vacuole, known as an inclusion. Although the EB was once considered metabolically inactive, quantitative proteomics reveal that EBs contain an abundance of proteins required for central metabolism and glucose catabolism 4,5. It is likely that these proteins are required for initial host cell entry and may help drive differentiation of EBs into RBs. Inside the inclusion, the EB transforms into an

RB, and begins the process of replication and nutrient acquisition6. After 24 hours, the RBs re- differentiate back into EBs and exit the host cell either by cell lysis or by extrusion of the inclusion, a process that leaves the host cell intact 7.

Serovar classifications

Strains of C. trachomatis are divided into three biovars that can be further subtyped into serovars. Biovars are variants that differ physiologically and/or biochemically from other strains of a particular species, and serovars are a subdivision of a species or subspecies that is distinguished by a characteristic set of antigens 8. The trachoma biovar contains serovars A-C and is the leading cause of non-congenital blindness in developing nations. The genital tract biovar contains serovars D-K and is the most prevalent sexually transmitted bacterial infection 9.

The final biovar, lymphogranuloma venereum (LGV), contains serovars L1-L3 and causes invasive urogenital or anorectal infection and is highly prevalent in HIV-infected men who have sex with men (MSM) 10-12.

2 Clinical manifestations

The genital tract biovar (serovars D-K) is transmitted through sexual contact with the penis, vagina, mouth or anus of an infected person. C. trachomatis can also be spread from an infected mother to her baby during child birth. In women, chlamydia infects epithelial cells of the endocervix and urethra resulting in a local inflammatory response that may cause redness and discharge (mucopurulent cervicitis) and urethritis. Despite infection initiating local inflammation, C. trachomatis infection is asymptomatic in 70-90% of women13. Because chlamydia infection in women can be asymptomatic and lacks abnormal physical examination findings, C. trachomatis can remain undetected in the genital tract for an extended period.

Untreated infections can lead to significant morbidity, including ascension of infection to the upper reproductive tract (i.e., uterus, Fallopian tubes), pelvic inflammatory disease (PID), chronic pelvic pain, and tubal infertility. Untreated infections have also been linked to adverse pregnancy outcomes such as ectopic pregnancy, intra-amniotic infection (chorioamnionitis), placental infection (placentitis), premature rupture of membranes, and preterm birth 14.

Additionally, infection can cause conjunctivitis and pneumonitis in some exposed newborns 15-17.

Diagnosis

Nucleic acid amplification tests (NAATs) are the most sensitive tests for detecting chlamydia and gonococcal infections18. NAATs can be performed on endocervical, urethral, vaginal, pharyngeal, rectal, or first-void urine samples. The accuracy of NAATs between sample types does not seem to vary significantly 18. Pooled dated from 29 studies show that 3 commercially available NAATs had a minimum sensitivity of approximately 80% for urine samples and 85.5% for cervical samples, and a minimum specificity of 99.1% for urine samples and 97.1% for cervical samples [16].

3 Until recently, the “gold standard” for the serological diagnosis of any chlamydia infection was the microimmunofluorescence (MIF) test originally developed by Wang and

Grayston for epidemiologic investigations of C. trachomatis and its involvement with trachoma

19. It was the only test that discriminated among species of chlamydia until the recent development of a peptide enzyme-linked immunosorbent assay 20,21.

Epidemiology

Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) and results in substantial morbidity and economic costs worldwide. The World Health

Organization (WHO) reported that in 2012, 131 million new cases of chlamydia occurred among adults and adolescents with a global incidence rate of 38 per 100 females and 3.3 per 100 males

22. The annual surveillance report released by the Center for Disease Control and Prevention

(CDC) has shown that during 2015 – 2016 the rates of chlamydia has increased by 4.7% from previous years. During 2012 – 2016, there was a total increase of 2.9 % in females and a 26.8 % increase in males 23. Reported cases of chlamydia are highest among people aged 15-24 and women 23. In 2017, the rates of reported cases of chlamydia were highest in Black women and in the South 23. Currently, there are limited chlamydia screening guidelines for males, which may underestimate the prevalence among young men 24.

Pelvic Inflammatory Disease

One manifestation of untreated C. trachomatis infection in women is pelvic inflammatory disease (PID). PID is a clinical syndrome that results from the ascension of pathogenic microorganisms from the cervix and vagina to the upper genital tract 25. If left untreated, PID can lead to infertility and permanent damage to a woman’s reproductive organs 26. PID is comprised of a spectrum of upper genital tract inflammatory disorders in women including any combination

4 of infection of the endometrium (endometritis), infection of the Fallopian tubes (salpingitis), tubo-ovarian abscess and pelvic peritonitis 25.

Several different microorganisms may cause or contribute to PID. Sexual transmission of

C. trachomatis and Neisseria gonorrhoeae has been implicated in 30-50% of PID cases 27,28.

Commensal microorganisms found at high levels in women with bacterial vaginosis (BV), have also been implicated in the pathogenesis of PID 29,30. Recent data suggests that Mycoplasma genitalium may also play a role in PID and may be associated with milder symptoms 31-34.

Because of the polymicrobial nature of PID, broad-spectrum treatment regimens that provide adequate coverage of likely pathogens are recommended.

Women with PID present with a variety of clinical signs and symptoms that range from subtle and mild to severe. When present, the most common symptoms of PID are lower abdominal pain, mild pelvic pain, cervical motion tenderness, increased vaginal discharge, fever, pain with intercourse, and painful and frequent urination. PID can go unrecognized by women and their health care providers when the symptoms are mild. Many women have a clinically silent spread of infection to the upper genital tract, which results in subclinical PID 25,35.

Subclinical PID, which may or may not advance to clinical PID, can result in long-term reproductive disability, including infertility, ectopic pregnancy, and chronic pelvic pain 35,36.

Despite lack of symptoms, histologic evidence of endometritis, which is associated with infection of the Fallopian tubes (salpingitis), has been demonstrated in women with subclinical

PID 35.

Screening and treatment of sexually active women for STIs such as chlamydia and gonorrhea have significantly reduced the incidence of PID in North America and Western

Europe 37-40. Despite recent progress, PID remains a problem because reproductive outcomes

5 among treated patients are still suboptimal. Subclinical PID remains poorly controlled, and programs aimed at its prevention are not feasible in much of the developing world 36.

Current treatment options

Chlamydia is curable with antibiotics. The WHO recommends either azithromycin or doxycycline as oral treatment options and these drugs are equally effective in eradicating infection 22. In the United States, there is a high prevalence of chlamydia infections in the general population, particularly among young women 41. Because of the large burden of disease and risk associated with infection in women, the CDC recommends annual chlamydia screening for all sexually active women younger than 25 years of age and women over 25 years of age with increased risk for infection (such as women with new or multiple sex partners) 9. Data from randomized controlled trials of chlamydia screening programs suggested that these programs can reduce the incidence of PID 42,43. Unexpectedly, widespread screening and treatment efforts have been accompanied by increased rates of reinfection. This has been attributed to a reduced duration of infection, which is hypothesized to arrest the development of protective adaptive immune responses (see “Adaptive Immunity”). Thus, a vaccine is essential for effective control efforts 44.

Innate immunity

Toll-like receptors

Toll-like receptors (TLRs) are a family of cell receptors that recognize pathogen associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), hypo-methylated

CpG-rich DNA, as well as double stranded and single stranded RNA. TLRs recognize microbial infection and have a critical role in the induction of innate and adaptive immune responses.

Signaling through TLR2 has been shown to be associated with increased oviduct pathology in

6 mice infected with C. muridarum, a natural respiratory pathogen for the mouse. When infected intravaginally, it infects the cervix and ascends to infect the upper genital tract, paralleling infection with C. trachomatis in women. Interestingly, although mice deficient in TLR2 display an infection course comparable to wildtype mice, TLR2-deficient mice do not develop significant pathology in the oviduct following C. muridarum infection 45. This suggests that engagement of TLR2 by chlamydia can act as a potential common pathway involved in both pathogen recognition and innate immunity and immunopathology. To date, no major chlamydial

TLR2 antigens have been specifically identified.

Chlamydial plasmid

Chlamydia trachomatis and C. muridarum contain a plasmid that has been found to play a significant role in chlamydial-associated pathology. In mice, C. muridarum organisms cured of their plasmid do not cause significant pathology in the upper genital tract 46. Moreover, plasmid- deficient C. muridarum mutants that retain the ability to infect the murine genital tract were unable to trigger TLR2-mediated signaling and did not develop oviduct pathology 47. These data suggest that a protein controlled by the chlamydial plasmid gene(s) may stimulate TLR2 signaling in cells, which can lead to oviduct pathology.

Matrix metalloproteinases

Matrix metalloproteinases (MMPs) are enzymes that play an important role in cellular turnover and extracellular matrix (ECM) remodeling in the female reproductive tract 48. MMPs are involved in the proteolysis and re-synthesis of the ECM 49,50, processing of chemokines and cytokines to active forms 51, the release of sequestered growth and signaling factors 52 and the chemotaxis and migration of leukocytes through inflamed tissues 53-55. Various members of the

MMPs, including MMP9, MMP10, MMP13, and others expressed by endometrial cells of mice

7 infected with C. muridarum have been implicated in tissue damage in the mouse endometrium and oviducts 56. Supporting the effect of MMPs in pathogenesis is the fact that use of an MMP inhibitor protected mice against chronic oviduct disease by C. muridarum 57. In humans, MMP9 expression has also been associated with trachomatous scarring 58,59. Additionally, expression of

MMP9 by human Fallopian tube cells infected with C. trachomatis was associated with tubal scarring 60.

Neutrophils

Human and animal studies of chlamydia infection reveal a direct correlation between neutrophil influx and development of tissue damage 61-66. One study that evaluated endometrial biopsies from women with suspected PID found that C. trachomatis infection was associated with an inflammatory milieu consisting of both acute and chronic leukocyte populations, including neutrophils, plasma cells, and periglandular lymphoid follicles containing transformed lymphocytes 67. Another study that evaluated women at risk for PID, determined that the presence of high levels of α-defensins, antimicrobial peptides produced by activated neutrophils, in the vagina were linked to endometritis in C. trachomatis-infected women 68.

Studies with the mouse model have supported observations in humans by demonstrating that enhanced or prolonged neutrophil influx into the oviducts is associated with the development of hydrosalpinx 66,69. In addition, mice deficient in CXCR2, a chemokine receptor for IL-8, display reduced acute inflammation and lower frequencies of hydrosalpinx, while strains of mice with elevated MIP-2, a neutrophil chemokine, exhibit worse disease 70,71.

Neutrophils likely contribute to pathology by releasing mediators that directly damage reproductive tract tissues. There is in vitro evidence for IL-1, a cytokine released by neutrophils and monocytes, to cause direct oviduct cell damage 72 and neutrophil release of the proteolytic

8 enzyme MMP9 has been implicated in the development of scarring and fibrosis of the murine oviduct after chlamydial infection 56,73. Further, infection of mice with a plasmid-deficient strain of C. muridarum cause minimal oviduct pathology despite a normal infection course 47. This reduced pathology is associated with reduced numbers of neutrophils in the oviducts on late days of infection 47. Overall, data for neutrophil involvement in the development of upper tract pathology is overwhelmingly supportive.

Cytokines and Chemokines

Several studies in animal models and in humans have shown that the first contact of C. trachomatis with host cells during infection leads to secretion of pro-inflammatory cytokines including TNFα, IL-8, IL-1α, IL-1β and GM-CSF 64,72,74,75. These cytokines, which are required to trigger the immune response, can cause collateral tissue damage 74-78.

IL-1

The Interleukin-1 family (IL-1 family) is a group of 11 cytokines that play central roles in the regulations of immune and inflammatory responses to infections or sterile insults. The most studied members of the family are IL-1α and IL-1β, which bind the same receptor (IL-1R). In chlamydia infection, studies in the mouse model and in vitro experiments indicate a role for IL-1 receptor (IL-1R) signaling in pathology. A direct role for IL-1 signaling and oviduct damage was observed in a human Fallopian tube organ culture model in vitro 72. In C. trachomatis infection, addition of the IL-1 receptor antagonist (IL-RA), which inhibits the IL-1 response, led to decreased tissue damage. This study also demonstrated that addition of IL-1α to the Fallopian tube organ culture could induce tissue damage 72even in the absence of chlamydia. IL-1 has also been shown to induce production of the neutrophil chemokine IL-8, which could further promote tissue damage in vivo by promoting the influx of neutrophils 72. Further, mouse studies of

9 chlamydia infection show that mice deficient in IL-1β 64 or IL-1R 79 have increased bacterial burden yet reduced levels of oviduct pathology, while mice deficient in IL-1RA have decreased bacterial burden and severe oviduct pathology. The cell types responsible for IL-1β production in the genital tract were determined to be neutrophils and macrophages 79, and IL-1β production by these cells is likely an important contributor to tissue damage.

The role of IL-1R signaling has not been well studied in humans. One study examining single nucleotide polymorphisms (SNPs) in IL-1β and IL-1RA detected no association with the development of tubal pathology in women with positive chlamydia serology 80. However, the authors only analyzed two SNPs in IL-1β as well as a number of tandem repeats in the second intron of IL-1RA 80.

TNFα

TNFα is a cytokine involved in systemic inflammation. It is mainly produced by activated macrophages, although it can be produced by CD4+ T cells, NK cells, neutrophils, mast cells, eosinophils, and neurons. Studies in the mouse model indicate that TNFα is not crucial for host defense against C. muridarum and promotes tissue damage in the genital tract.

TNFα -/- mice resolve infection normally, and oviduct pathology is significantly reduced 81.

TLR2-/- mice or mice infected with a plasmid-cured strain of C. muridarum, CM3.1, develop less severe oviduct pathology despite normal resolution of infection, and this reduced pathology was associated with significantly decreased TNFα in the genital tract 45,47. Other studies have determined that TNFα production by CD8+ T cells contributes to pathology in this model 81.

These CD8+ T cells were further shown to be chlamydia-specific TNFα producing cells 82.

Polymorphisms in the TNFα promoter have been correlated with disease in humans. In a case-control study of individuals from The Gambia, possession of the TNF-308A allele resulted

10 in increased TNFα production from peripheral blood mononuclear cells (PBMCs) in response to

EB stimulation and a significantly increased risk of trichiasis 83. Additionally, the TNF-308A allele was also associated with an increased risk of severe Fallopian tube damage in women with tubal factor infertility (TFI) associated with C. trachomatis infection 84.

IL-17A

Interleukin 17A (IL-17A) is a proinflammatory cytokine produced by T helper cells

(known as T helper 17 cells) in response to stimulation with IL-23. Promoting inflammation, IL-

17A acts in concert with TNFα and IL-1. IL-17A, and other variants, recruit immune cells such as monocytes and neutrophils to the site of inflammation. The role of Th17 cells in chlamydial pathogenesis has been addressed by serval studies 85 and association between IL-17A and chlamydial disease has been suggested from animal models as well has humans 86. Studies in the mouse model, examining the role of IL-17A in protective immunity, have had conflicting results.

IL-17A is critical for host defense against C. muridarum pulmonary infection. Resolution of infection, prevention of bacterial dissemination, and the development of Th1 immunity were compromised in the absence of IL-17A-mediated signaling in this model 87,88. This protective role was due to the ability of IL-17A to induce IL-12 production by dendritic cells 87. However, these studies did not evaluate chronic disease sequelae. Using IL-17A knockout mice on a

C57BL/6 wild-type background, IL-17A was shown to be pro-atherogenic in a high fat diet- induced C. pneumoniae-accelerated atherosclerosis in mice, providing evidence for a pathogenic role of Th17 cells in disease 89. This was supported by evidence using the model of genital tract infection which demonstrate that IL-17A knockout mice on either BALB/c or C57BL/6 wild- type background displayed reduced infection clearance and oviduct pathology 90,91. Other studies have found that IL-17 receptor A knockout mice displayed reduced neutrophil recruitment and

11 Th1 responses, but no change in the course of infection or oviduct pathology 92. Meanwhile, mice deficient in IL-23, which is required for activation of IL-22 and Th17 cells, showed no difference in the Th1 response or resolution of infection, but had decreased Th17 cells and a slight reduction in oviduct pathology 93. Elevated levels of IL-17A have been associated with increased disease and enhanced neutrophil recruitment 94,95.

The role of IL-17A in humans has not been extensively studied. In one study, expression of IL-17A and the genes S100A7 and CXCL5 was associated with the presence of trachomatous conjunctival inflammation in children in Tanzania 96. Another study of women infected with C. trachomatis detected IL-22 and IL-17 production by CD4+ T cells isolated from cervical washes

97, but no correlation was made between the presence of these cells and disease. In men, increased seminal fluid levels of IL-17A may be associated with C. trachomatis pathogenesis 98.

Overall, chlamydia infection upregulates many innate inflammatory mediators within the reproductive tract, and the presence of these mediators have been associated with disease. The interactions of these mediators and how inflammatory responses might be modified therapeutically, to limit tissue damage, require further study.

Chemokines

Chemokines are cytokines that can induce directed chemotaxis in nearby responsive cells.

Since chlamydia is an infection localized to the genital tract, cells involved in bacterial clearance must be able to traffic to the site of infection. Many studies have demonstrated patterns of either

Th1 or Th2-associated chemokines in diseased tissues previously shown to contain large infiltrates of either Th1 or Th2 cells99. For instance, in the Th1-mediated disease multiple sclerosis, high levels of chemokines CXCL10 (interferon-inducible protein 10 [IP-10]), CXCL9

(monokine induced by gamma interferon [MIG]), and CCL5 (RANTES) are found in the

12 cerebrospinal fluid, suggesting that chemokine profile plays a central role in determining the predominate T-cell subset associated with a particular disease or infection100. Some chemokines are known to be associated with disease in chlamydia infection. In women, the interferon- induced chemokines CXCL10 and CXCL11 have been associated with C. trachomatis related infertility101. In mice, chlamydia infection leads to the production of a type I interferon response, which can induce the production of chemokines CXCL9, CXCL10, and CXCL11. Type I interferon signaling in mice has been associated with exacerbated disease. Mice deficient in type

I interferon signaling have higher infiltrates of CD4+ T cells, reduced chlamydial shedding, less pathology, and decreased levels of CXCL10 in genital secretions102. Recently, it has been shown that expression of CXCL9, CXCL10, and CXCL11 were associated with upper genital tract pathology in chlamydia infected mice 103. This pathology was associated with increased recruitment of non-protective T cells to the site of infection.

Adaptive immunity

Humoral immunity

Immunoglobulins (Igs) play important roles in adaptive immunity. They have several mechanisms by which they can kill pathogens including neutralization, agglutination, and activation of complement. Antibodies can also enhance the cellular-mediated immune response in a process known as antibody-dependent cell-mediated cytotoxicity. Antibodies can be transported by mucosal epithelia during infections. IgA and IgG are the dominant Ig isotypes found in mucosal secretions 104, however IgG is the major antibody subclass in genital secretions, where it predominates over IgA 105. In murine chlamydia infection, B cells and antibodies have mostly been associated with protective immunity, particularly to secondary infections, rather than pathogenesis 106-109. In fact, it has been shown that the absence of B cells enhances oviduct

13 pathology 110. Additionally, it has been shown that B cells are important mediators in preventing the dissemination of chlamydia into the peritoneal cavity 111. Data from mouse and guinea pig models link serum anti-chlamydial IgG to reduced bacterial load, duration of infection, and pathology 112,113, while data from human studies indicate minimal to no role for serum anti- chlamydia IgG in resistance to reinfection.

IgG in human studies

In humans, high titers of C. trachomatis-specific antibodies do not correlate with resolution of infection, and in fact, are more strongly correlated with increased severity of sequelae of infection, such as tubal infertility in women 114. High antibody titers may serve as a marker of extent of infection or exposure. In women with fertility disorders, significant prevalence of antibody to heat shock proteins 10 and 60 (hsp10, hsp60) have been reported 115.

A previous study using a microimmunofluorescence (MIF) assay for IgG to chlamydia elementary bodies revealed that although higher titers of anti-chlamydia IgG were associated with reduced cervical chlamydia burden, titers failed to correlate with reduced chlamydial ascension to the endometrium or reduced risk for reinfection 116.

IgA in mouse models

Female mice deficient in IgA show no defects in their ability to clear primary or secondary chlamydia infections when compared to wild-type mice, suggesting a dispensable role for IgA 112. However, serum IgA titers to MOMP have been shown t to be associated with reduced chlamydia burden, magnitude, and duration of infection in female mice immunized with

MOMP, and in immunized male mice 117-119.

IgA in human studies

Data from human studies indicate that serum anti-chlamydia IgA is associated with reduced bacterial ascension in postabortal women 120. Another study purports that serum IgA has minimal to no role during chlamydia infections 121. However, since chlamydia infection is localized to the reproductive tract, locally produced antibody responses may be more informative than serum. Indeed, human data from chlamydia-infected women demonstrate that cervical IgA is inversely correlated with bacterial burden 121. Unpublished data from our lab also supports these findings. Generally, antibodies appear to play a supportive, yet non-essential, role.

Cell-mediated immunity

CD4+ T cells

CD4+ T cells and protection in mice

Earlier studies identified a critical role of T cells in chlamydia infection, with subsequent studies highlighting the importance of CD4+ T cells in protective immunity. CD4+ T cells are critical for protection against chlamydia in the mouse model of genital tract infection. It was shown in some of the first studies characterizing the mouse model of Chlamydia muridarum infection that athymic nude mice, which lack B and T cells, were unable to resolve infection 122.

Another early study showed that severe combined immunodeficiency (SCID) mice, which lack functional B and T cells, exhibited minimal clearance of infection from the genital tract, and disseminated infection was common 123. The T cell receptor (TCR) is important for recognizing fragments of antigens. One study showed that TCRβ chain deficient mice could not resolve infection while TCRδ chain deficient mice resolved infection normally 124. The specific role of

CD4+ T cells was demonstrated in a study where MHC Class II-/- and CD4-/- mice exhibited significantly delayed resolution of infection 125. During secondary genital tract infection,

15 depletion of CD4+ T cells from C57BL/6 or B cell (µMT-/-) deficient mice was shown to significantly delay resolution of infection 126.

CD4+ T cells and protection in humans

Subsequent studies identified the role of CT-specific CD4 Th1 cell immunity against CT.

A study examining the PBMC responses of commercial sex workers in Nairobi, Kenya determined that production of IFN in response to hsp60 was associated with reduced risk of CT reinfection 127. A subsequent study revealed that isolated CD4+ T cells produced at a significantly higher frequency than isolated CD8+ T cells in response to EBs and hsp60-1 antigen 128. The protective role of T cell responses to chlamydia hsp60 was supported by a study in Australia. Women presenting with their first C. trachomatis infection and uninfected women exhibited similar levels of IFN production by PBMCs in response to hsp60, but levels were significantly reduced in women with repeated infections or with chlamydia-induced PID 129.

Recently, IFN production by CD4+ T cells stimulated with EBs and major outer membrane protein (MOMP) peptides has been shown to be associated with protection from reinfection 130.

For women with HIV and chlamydial genital tract infection, the risk of developing PID was inversely correlated with the number of CD4+ T cells in the peripheral blood 131. A CD4+ T cell count of less than 400 was associated with a 21.7- fold increased risk of developing chlamydia- induced PID 131. Notably, there was no association between the CD8+ T cell count and risk of

PID 131. Recently, cohort studies in chlamydia-infected women, have determined that antigen- specific IFN -producing CD4+ T cells are associated with protection 132,133.

Peripheral blood responses to chlamydial antigens have also been detected in individuals from trachoma endemic areas. A study of individuals from The Gambia with and without trachomatous scarring determined that stimulation of PBMCs with MOMP or EBs resulted in

16 increased levels of IFN production relative to unstimulated samples in both groups, but only individuals without scarring had detectable levels of IFN in response to hsp60 134, suggesting a protective role for IFN against disease. In this study, significant associations were detected between specific MHC class II alleles and the strength of CT-specific immune responses. In the participants, the HLA-DRB1*11 allele was associated with increased IFN production in response to EBs, whereas those expressing the HLA-DRBI*1304 allele exhibited increased T cell proliferation in response to hsp60 stimulation 134. An additional study examining the peripheral blood responses of children in The Gambia detected increased proliferation of PBMCs in response to stimulation with EBs, MOMP, and hsp60 in children that had recently resolved their trachomatous inflammation relative to those with continued inflammation 135.

CD4+ T cells and pathology in mice

Studies in mice indicate that the magnitude of the CD4+ T cell IFN response is inversely correlated with the development of oviduct pathology. MHC Class II-/- mice 125 and IFN-/- mice 92 infected with chlamydia develop severe oviduct pathology, which is likely due to decreased bacterial clearance in these mice. C57BL/6 mice exhibit a more robust CD4+ T cell

IFN response and decreased IL-10 production relative to C3H/HeN or BALB/c mice, and this is associated with faster resolution of chlamydia genital tract infection and lower levels of oviduct pathology 45,69. IL-10-/- mice rapidly induced a chlamydia specific CD4+ T cell IFN response and exhibited faster resolution of primary infection, resistance to challenge infection, and dramatically reduced oviduct pathology 136. Transfer of CD4+ T cells specific for chlamydial protease-like activating factor (CPAF) that can produce IFN resulted in faster resolution of infection and prevention of oviduct pathology 137. Upon challenge infection, a decreased frequency of neutrophils and an increased frequency of IFN producing T cells in the genital

17 tract have been associated with reduced levels of ascending infection and decreased pathology

138. A renewed susceptibility to genital tract infection after resolution of primary infection coincides with the departure of chlamydia specific T cells from the genital tract 139.

CD4+ T cells and pathology in humans

In an additional study from Nairobi, Kenya, in women seeking treatment for infertility, the presence of specific MHC class II alleles was associated with infertility and C. trachomatis seropositivity 140. HLA-DQA*0101 and HLA-DQB*0501 were associated with an increased risk of C. trachomatis associated infertility and HLA-DQA*0102 was associated with a decreased risk 140. The interaction between these alleles and CD4+ T cell responses to chlamydia was not examined.

Induction of IL-10 producing CD4+ T cells, possibly regulatory T cells, following CT infection has been associated with increased pathogenicity. A study of infertile women found that stimulation of their T cells with chlamydia hsp60, resulted in production of IL-10, a cytokine produced by regulatory T cells (Tregs) 141. In patients with conjunctival chlamydial infection, conjunctival IL-10 and FoxP3 transcripts were elevated, and in patients where clinical signs of disease were present, but no detectable infection, FoxP3 transcript levels were elevated 142.

CD8+ T cells

CD8+ T cells and protection in mice

Like neutrophils, CD8+ T cells have been shown to possess anti-chlamydial activity.

Some clones of CD8+ T cells have been shown to induce protective immunity against chlamydial infection, but they do so through their ability to secrete IFN, not cytolysis 143,144. In the mouse model, a deficiency in CD8+ T cells does not impair infection control. Mice deficient in expression of MHC class I molecules (βeta2microglobulin-/- mice; TAP1-/- mice) exhibit

18 normal resolution of both primary and challenge infection with C. muridarum 81,125. Mice deficient in CD8 or treated with anti-CD8 antibody also resolve infection normally 81, as do

C57BL/6 and antibody deficient mice (µMT-/- mice) depleted of CD8+ T cells upon challenge infection 112. Chlamydia infection upregulates molecules such as PD-L1 at the site of infection.

Depletion or inhibition of PD-L1 has been shown to enhance CD8-mediated clearance of infection 145. In addition, mice deficient in pathways used by CD8+ T cells to lyse target cells including perforin 81,146, Fas ligand (FasL), and both perforin and FasL resolve infection normally without exhibiting a compensatory cytokine response 146. Lastly, adoptive transfer of

CD8+ T cells isolated from the spleens of mice that had resolved both primary and secondary genital tract infection with C. muridarum conferred no protection when transferred into immunologically normal mice prior to infection 147.

CD8+ T cells can provide a limited degree of protection in specific adoptive transfer models. CD8+ T cell clones isolated from the spleens of mice after intravenous (i.v.) injection of

C. muridarum have been demonstrated to promote resolution of infection when transferred into intravaginally infected nude mice and to produce both IFN and TNFα in a chlamydia-specific

147 manner. In two separate studies, CD8+ T cells isolated from the spleen of mice infected either intraperitoneally (i.p.) or intravenously (i.v.) with C. trachomatis serovar L2 were protective upon subsequent i.v. infection with the same strain of chlamydia, and this protection was IFN dependent 143,148.

CD8+ T cells and protection in humans

Until recently, there has been no known role for CD8+ T cell responses in protection in humans. In a recent cohort study of both men and women, IFN production by CD4+ and CD8+ peripheral blood T cells were measured after co-culture with antigen-presenting cells (APCs)

19 preincubated with recombinant Escherichia coli expressing chlamydia antigens. The results indicated that distinct CD8+ T cells were associate with an effective immune response, defined as an infection that cleared without treatment or absence of infection despite documented exposure 132. In a similar study, our lab examined samples from a cohort of women at high risk for chlamydia infection, and determined that higher frequencies of peripheral blood CD8+ IFN- responses to chlamydial antigens were significantly associated with reduced ascending infection

133. For some antigens, the frequency of CD8+ T cell responses was higher in women with infection limited to their cervix, than in women with endometrial infection 133.

CD8+ T cells and pathology in mice

Studies in the mouse model also indicate that CD8+ T cells contribute to the development of chlamydia-induced immunopathology. Mice deficient in CD8+ T cells or perforin develop reduced levels of oviduct pathology upon intravaginal infection with C. muridarum 81,146,149.

Adoptive transfer studies revealed that CD8+ T cell production of TNFα was a significant mediator of oviduct pathology and did not significantly contribute to host defense 81. OT-1 transgenic mice, which recognize ovalbumin peptide instead of chlamydial antigens, were infected with C. muridarum. Vaginal chlamydial shedding in these mice were normal and oviduct pathology was significantly reduced 150. However, repletion of OT-1 mice with wild-type

CD8+ T cells restored the ability of these mice to generate chlamydia-specific CD8+ T cells and induce pathology82. It has been further demonstrated that TNF receptor 2 on CD8+ T cells has been shown to be responsible for the activation of chlamydia-specific T cells and the causation of pathology 151. In mice deficient in CD8, there was significant protection against chlamydia- induced infertility following four episodes of infection 152. These results suggest CD8+ T cells cause genital tract pathology and indicate TNFα production as a pathogenic mechanism. Indeed,

20 it has recently been demonstrated that C. trachomatis serovar D induces infertility in wildtype, but not TNFα, knockout female mice. Reconstitution of wild-type T cells into the TNFα knockout mice restored infertility 153.

CD8+ T cells and pathology in humans

The association between CD8+ T cell responses and disease resulting from chlamydia genital tract infection has not been extensively examined in humans. In a study of commercial sex workers in Nairobi, Kenya, the MHC class I allele HLA-A31 was associated with the development of chlamydia-induced PID in women with documented chlamydia infection 131. The correlation of this allele with CD8+ T cell function was not determined.

Examination of the correlation between immune responses and disease is more feasible in regions where ocular infection with chlamydia is endemic. Almost all the individuals in the population are exposed to chlamydia, but only some individuals develop disease. For example, in a study of individuals from The Gambia with and without trachomatous scarring, approximately

90% of individuals in both the case and control groups had detectable antibody to C. trachomatis

154. In this study, it was determined that individuals with the MHC Class I allele HLA-A*6802 were 3 times more likely to develop scarring than individuals without this allele. In contrast, the

MHC II genes HLA-DRB1 and HLA-DQB1 were not significantly associated with disease.

Interestingly, in a follow-up study, chlamydia-specific CD8+ T cell responses were not detected in the peripheral blood of individuals with and without trichiasis or trachomatous scarring that possessed the HLA-A*6802 allele. This is likely because only responses to predicted epitopes in the proteins MOMP, macrophage infectivity potentiator (MIP), and heat shock protein 70

(hsp70) were examined, but responses to infected cells were not examined 155. In another study of peripheral blood responses of individuals from The Gambia with HLA-B8 and HLA-B35

21 alleles, cytolytic responses were detected in response to predicted MOMP and heat shock protein

60 (hsp60) peptides in 6 of 26 people examined, and responses were only detected for CD8+ T cells isolated from children that were in the process of resolving infection or adults without scarring. No responses were detected for the PBMCs of adults with trachomatous scarring 156. A later study using tetramers to examine HLA-A2 specific responses to MOMP peptides in the peripheral blood of children from The Gambia, revealed an association between positive tetramer staining and the presence of active infection or repeated episodes of infection 157. No association was found between tetramer binding and the presence of clinical symptoms 157. Other studies involving Tanzanian and Gambian populations, found that MHC I genes HLA-B*07 and HLA-

B*08 were associated with trichiasis or trachomatous scarring 158,159. Recently, human studies that evaluated antigen-specific CD4+ and CD8+ T cell responses in the peripheral blood of chlamydia-infected men and women, determined that distinct antigen-specific IFNγ-producing

CD8+ T cell responses were associated with an ineffective immune response or tubal factor infertility (TFI) 132. In this study, an ineffective immune response was defined as having sustained persistent infection, repeat infection, or diagnosis of clinical PID.

Summary of immune responses associated with chlamydia-induced pathology

C. trachomatis is not a bacterium that secretes toxins to cause cellular damage. Instead, the immune response induced by chlamydia infection drives host pathology 160. Animal models of ocular and female genital infection reveal a direct correlation between neutrophil influx and development of tissue damage 61-66. In addition, human transcriptional profiling and genetic studies have determined an association of enhanced innate proinflammatory responses with trachomatous scarring 66,161,162. Finally, there is in vitro evidence for IL-1, a cytokine released by neutrophils and monocytes, to cause direct oviduct cell damage 72.

22 Since the innate inflammatory response is induced by the interaction of pathogen associated molecular patterns (PAMPs) with pathogen recognition receptors (PRRs) on innate inflammatory cells and host epithelial cells, it should not be surprising that increased bacterial burden leads to enhanced inflammation and disease 62,163,164. The mouse model of C. muridarum genital infection revealed that repeated infections that were abbreviated by antibiotic treatment led to protection from oviduct disease that was associated with a significant reduction in frequency of neutrophils and an increase in the frequency of T cells infiltrating the genital tract upon challenge 138. Furthermore, a single infection with a plasmid-deficient strain of C. muridarum, protects mice from oviduct disease upon challenge with the fully virulent parental strain 47. This protection is associated with reduced neutrophil influx and an anamnestic T cell response 138. Thus, avoidance of chlamydial-induced neutrophil influx and neutrophil activation appears essential for disease prevention.

Human epidemiological studies demonstrate an increased risk of disease with recurrent infections 131,165. However, the contribution of pathological effects of the primary infection versus subsequent infections is unknown, and each successful infection would induce an element of tissue-damaging innate responses. Interferon-γ and IL-12 mediate protective T-helper 1 (Th1) responses124, while T-helper 2 (Th2) responses have been shown to be non-protective 134. CD8+

T cells have been shown to play a role in pathogenesis in the macaque and mouse models of genital tract infection, possibly through the production of TNFα 81,82,166. Currently, there is no evidence for the role of B cells in tissue pathology during chlamydia infection.

23 Current study

INTRODUCTION

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. Chlamydia infection is often asymptomatic, which results in individuals going undiagnosed and untreated for extended periods of time. In women, at least 20 to 40% of untreated cervical chlamydial infections ascend to the upper genital tract 167, leading to inflammation of the uterus and Fallopian tubes. This can cause reproductive health complications such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and infertility. Since women who have had upper genital tract infection and inflammation are at greater risk of repeat disease and eventual infertility 35,68, biomarkers of disease risk are urgently needed to enable enhanced targeted screening in women at greatest risk for disease. Currently, a major research gap in the field, is the lack of a noninvasive biomarker that can accurately identify women with mild or asymptomatic upper genital tract infection, which can result in subclinical PID. Since chlamydia does not disseminate, the biomarkers of disease may be localized to the site of infection. Thus, an important goal of our research is to identify cytokine profiles in cervical secretions of women with and without upper genital tract chlamydia infection to determine immune responses associated with disease. Identification of cervical cytokines associated with altered susceptibility to chlamydial ascension or reinfection could provide clues regarding harmful or protective immune responses and instruct novel therapeutics and vaccine design. Biomarker development could guide targeted screening of women at increased risk for upper reproductive tract pathology and serve as a surrogate endpoint in vaccine trials.

Considerations for designing an effective biomarker require a delineation of immune responses associated with protection and those associated with pathology. Immunoepidemiologic

24 studies in women indicate CD4+ T cells are important in protection 127,130-133. In women with

HIV infection, lower CD4+ counts were found to be a risk factor for chlamydia-induced PID 131.

Among female sex workers, peripheral blood mononuclear cell IFN- responses to chlamydial antigens were associated with reduced reinfection 33.

Previously, using samples collected from 251 generally healthy, highly sexually active young women in Pittsburgh enrolled in a T cell Response Against Chlamydia (TRAC) study, we performed multiple analyses of factors associated with protection or susceptibility to chlamydia infection. T cell studies in a subset of participants revealed higher frequencies of peripheral blood CD4+ and CD8+ IFN- responses to chlamydial antigens were associated with reduced incident and ascending infection, respectively 133. Analysis of clinical and behavioral factors in the entire cohort revealed oral contraceptives and gonorrhea were associated with endometrial infection; and reduced age, gonorrhea, cervical chlamydial infection at enrollment, and sexual exposure to new, uncircumcised or infected male partners significantly increased risk of incident infection 133. Analyses of serum and cervical secretion antibody responses within the first 150 women enrolled in TRAC revealed that although serum and cervical anti-chlamydia IgG and cervical anti-chlamydia IgA levels correlated inversely with cervical burden, the burden lowering effects were insufficient to prevent ascension. Further, longitudinal analyses revealed that serum and cervical anti-chlamydia IgG correlated with significantly increased risk of reinfection during a year of follow-up 168.

Since chlamydia predominantly infects mucosal epithelial cells, the mucosal immune response, including cervical cytokine secretion, is likely to play an important role in immunity.

Many cytokines can been detected in the endometrial epithelium of uninfected women including

CCL3 (MIP-1α), CCL5 (RANTES), CCL2 (MCP-1) and CCL11 (Eotaxin) 169,170. Additionally,

25 limited studies have compared a small number of cytokines between chlamydia infected and uninfected women in either endocervical, cervicovaginal, or vaginal secretions as well as in vitro in chlamydia-infected polarized endocervical epithelial cells 77,171-174. One study determined that expression of the chemokine CXCL13 (B cell chemoattractant) was associated with C. trachomatis infection of the female genital tract 175. A study by Arno et al., determined that women who were culture positive for chlamydia at the endocervix had significantly higher levels of IFN- than culture negative patients; no correlation was found between IFN- levels and numbers of organisms 172. Detection of chlamydia infection among 396 adolescent and adult women, ~60% of whom were HIV-infected, was associated with lower IL-2 and higher IL-12 levels compared to uninfected women 176. Finally, in a small cohort of New Delhi, Indian women, IFN-, IL-12, IL-1, and IL-10, were increased in those positive for chlamydia infection

177. None of these studies correlated cytokines with disease risk.

In mice, cytokine secretion in the cervix may regulate the recruitment of chlamydia- specific lymphocytes to the genital tract 178. For example, C. trachomatis infection can induce pro-inflammatory cytokines such as interleukins 1, 6, 8 (IL-1, IL-6, IL-8), tumor necrosis factor- alpha (TNFα), and colony stimulating factor 2 (CSF 2) 75 which recruit immune cells such as natural killer cells and phagocytes. Following an established intracellular infection, the T-cell mediated immune response becomes the critical element required for clearance 179. Further, mice have been shown to have differential expression of chemokines in the upper and lower genital tract in response to chlamydia infection 180. Expression of CXCL9, CXCL10, and CXCL11 have been shown be associated with upper genital tract pathology in mice 103.

As demonstrated by mouse models and human studies, cell-mediated immune responses are essential for protective immunity to chlamydia. T cell-mediated eradication of chlamydia

26 within the endocervical epithelium requires T cells be present at this infected mucosal site. This can occur either through the induction of tissue resident memory T cells, or trafficking of chlamydia-specific T cells from the peripheral blood to the endocervix. Both processes necessitate local production of chemokines and cytokines 181-184. We hypothesized that a comparative analysis of a broad array of cytokines in situ in women infected with chlamydia would reveal increased levels of cytokines that recruit and/or activate protective T cells in women with infection limited to their cervix compared to those in whom the organism has ascended to the uterus, and that T cell promoting cytokines would be increased at baseline in infected women who remained uninfected versus those who became reinfected during a year of follow-up. We used multivariable stepwise regression analysis and adjusted for the confounder of cervical bacterial load, as well as behavioral and clinical confounders previously identified in this cohort.

Analysis of 49 inflammatory proteins revealed that CXCL10, TNFα, and CXCL13 were associated with significantly increased odds of endometrial infection, while IL-16 was associated with significantly decreased odds. VEGF and IL-14 were associated with decreased and increased risk of reinfection after univariable analysis but were excluded from the final multivariable model after adjusting for clinical and behavior covariates. These data reveal proteins in cervical secretions that could be tested as predictive biomarkers of risk in future patient cohorts.

METHODS

Study population

The Institutional Review Boards for Human Subject Research at the University of

Pittsburgh and the University of North Carolina approved the study and all participants provided

27 written informed consent prior to inclusion. Samples were collected from participants recruited into a previously described T cell Response Against Chlamydia (TRAC) cohort 116, which was comprised of 246 asymptomatic women (age 15-30 years) at high risk for sexually transmitted infections from three urban sites in Pittsburgh, Pennsylvania from 2011-2015. Eligibility criteria included clinical evidence of mucopurulent cervicitis, diagnosis of chlamydia prior to treatment, or reported sexual contact with an individual recently diagnosed with chlamydial urethritis or nongonococcal urethritis. Women diagnosed with clinical pelvic inflammatory disease (PID) were excluded.

Demographic and clinical data were obtained using standardized questionnaires regarding behavioral practices, sex exposure, contraceptive methods, and symptoms. Cervical and endometrial swab samples were obtained for microbiologic testing 116. Cervical secretions were collected by placement of an ophthalmic Merocel® sponge (Beaver-Visitec International Inc.,

Waltham, MA) within the distal endocervix of each patient for 30 seconds. The retrieved sponge was placed into a cryovial on ice until transfer to the lab where the vials were stored in liquid nitrogen. Serum and peripheral blood mononuclear cells (PBMCs) were obtained for antibody and T cell assays. Participants returned for cervical infection screening and serum and PBMC collection at 1, 4, 8, and 12 months after enrollment. A standard questionnaire eliciting the participants’ interim medical and sexual history, and exposure were gathered at each follow-up visit. chlamydial cervical burden for participants was estimated via quantitative PCR using genomic DNA extracted from reserved cervical swab eluates as template 116.

Definition of clinical and microbiological subgroups

Women were assigned to groups according to the extent of their infection at enrollment.

Uninfected women were those who had a negative cervical swab for chlamydia by nucleic acid

28 amplification testing (NAAT). In infected participants, women with a chlamydia-positive cervical swab and chlamydia-negative endometrial sample were defined as Endo-. Women who tested positive for chlamydia at both the cervix and the endometrium were defined as Endo+.

Among 246 women analyzed, 85 (35%) were uninfected, 92 (37%) had lower tract infection

(Endo-), and 69 (28%) had upper tract infection (Endo+). No participant tested positive solely in the endometrium.

Additionally, women were grouped based on their infection status throughout follow-up.

Women who had a negative cervical swab for chlamydia by NAAT at 4 follow visits and never reported having an infection between visits were defined as follow-up negative. Women who tested positive for chlamydial infection at any follow-up visit or who reported having an infection between visits that was diagnosed at an outside clinic comprised the follow-up positive group. Within these groups, 95 (38%) women where follow-up negative, and 66 (28%) were follow-up positive. Women who did not attend at least 4 follow-up visits were determined to have an unknown infection status (N=85, 35%).

Quantification of cytokines in cervical secretions

Data acquisition. Cervical secretions collected at enrollment were eluted for protein assays as described with slight modifications 185. The cryovials and ophthalmic sponges were weighed to estimate the volume of secretions absorbed onto the sponge. Corning® Costar® Spin-

X® centrifuge tubes containing 0.45 μm filters (Millipore Sigma, St. Louis, MO) were equilibrated with 500μl of blocking buffer (PBS, 2% BSA, and 0.05% Tween-20) for 30 minutes at room temperature. Filters were then washed 3 times with 100μl PBS. Sponges were equilibrated using 300μl of elution buffer (PBS, 0.5% BSA, 0.05% Tween-20, and protease inhibitor) before being placed in Spin-X tubes where they were incubated on ice for 10 minutes.

29 Spin-X tubes containing sponges were centrifuged at 10,000 rpm for 1 hour at 4°C and eluted secretions were stored at -80°C. A dilution factor was calculated based on the estimated volume of the secretion and pre- and post- weight of the collection tube: ((x-y)+ 0.36g of elution buffer)/

(x-y), where x equals the weight of the sponge + cryovial tube and y is the average weight of the dry cryovial, based on independently weighing three dry cryovials. Cytokine levels were determined using Milliplex Magnetic Bead Assay Kits (Millipore Sigma, St. Louis, MO) at the

Duke University Immunology Core Lab, according to manufacturers’ instructions, using a

Luminex® fluorescence reader (Millipore Sigma, St. Louis, MO). Kits included lyophilized standards that were reconstituted and diluted at 7 serial concentrations following manufacturers’ instructions for generation of standard curves. Standards included all recombinant cytokines tested and were considered as positive controls for the procedure.

Expression of 57 cytokines in cervical sponge eluates of the 246 women were measured using customized panels across four different kits: (i) Milliplex Map Human

Cytokine/Chemokine Magnetic Bead Panel-Premixed 41 Plex-Immunology Multiplex Assay, (ii)

Milliplex Map Human Cytokine/Chemokine Magnetic Bead Panel II- Immunology Multiplex

Assay, (iii) Milliplex Map Human Cytokine/Chemokine Magnetic Bead Panel III- Immunology

Multiplex Assay, and (iv) Milliplex Map Human Cytokine/Chemokine Magnetic Bead Panel IV-

Immunology Multiplex.

Data processing. Cytokines with out of range (< LLOQ or >ULOQ) measurements over

60 percent were excluded (N=8). Forty-nine cytokines remained for further analysis after data exclusion. Missing cytokine data were imputed by mean imputation, which substitutes the missing expression value of a cytokine with the average expression of all available samples in the same group. The expression data was log2 transformed to improve normal distribution.

30 Because all samples were not tested at the same time, there may be differences in cytokines secretion that were not due to biological differences (e.g. temperature, reagents, time of day when the assay is done etc.). A single reference sample was included in each batch for quality control and to measure batch effect. We corrected for batch effect using the ComBat method 186.

The Combat method measures the mean and variance for each cytokine across batches and adjusts for these differences.

Statistical Analysis

Preliminary statistical analysis

Initial analysis to determine the association between cytokine secretion and bacterial ascension was conducted using GraphPad Prism 7 and RStudio. I performed a univariable analysis that was a logistic regression. Data were log2 transformed prior to analysis. The model was adjusted for hormonal birth control (OCPs), co-infection with Neisseria gonorrhea and

Mycoplasma genitalium. In the initial analysis, cervical bacterial burden was a categorical variable. The median of cervical bacterial burden was 3.71x104 DNA copies/swab. Women with a cervical bacterial burden greater than the median were classified as “High burden” and women with a cervical burden less than the median were classified as “Low burden”. Based on these definitions, 33% (N=80) of women had a high burden and 33% (N=81) of women had a low burden. Uninfected women did not have a cervical bacterial burden (N=85, 34%). In an initial analysis restricted to women with high bacterial burden, some significant results were obtained

(Table 1). These findings prompted discussions with an expert biostatistician who performed all analyses moving forward. In subsequent analysis, cervical bacterial burden was changed from a categorical variable to a continuous variable. Although, M. genitalium has been associated with

PID, it was not significant in these analyses, and therefore was not adjusted for in subsequent

31 analyses. Results of my initial univariate analysis are recorded in Table 1. Results of the second univariable analysis performed by the biostatisticians are recorded in Table 5. Results between the first and second analysis showed some consistency. It appears the adjustment for M. genitalium and the shift of bacterial burden from a categorical variable to a continuous variable did not alter the results very much. Most cytokines that were associated with increased risk of endometrial infection in the initial analysis remained associated with increased risk in the secondary analysis (CXCL10, CXCL9, IL-17A, TNFα, CXCL13). Additionally, the cytokines that were associated with decreased risk for endometrial infection in the preliminary univariable regression analysis remained associated with decreased risk (PDGF-AA, IL-15, CXCL14,

PDGF-BB, IL-16, FGF-2, IL-14). The most noticeable difference between the two analyses is the adjustment for batch effect caused some variance in the adjusted odds ratio and/or p value.

As a result of batch correction, cytokines from the initial analysis had increased or decreased significance. The correction for batch effect helped to delineate the differences between the groups due to biological differences not technical ones. All results of the preliminary analysis are recorded in Table 1. The following methods and results detailed in the thesis reflect analyses performed by Dr. Xiaojing Zheng and her students Wujuan Zhong and Li Dong, with my support.

Relationship between cervical cytokines and endometrial infection.

We log2 transformed cytokine concentrations and log10 transformed chlamydia 16S genome copies. We then performed Shapiro–Wilk normality tests to ensure normal distribution.

The correlation of cytokines with cervical chlamydia burden was conducted by Pearson's correlation test. We then determined the association of cytokines with ascending infection

(Endo+ versus Endo-) by univariable logistic regression, that measured one independent variable

32 at a time, with adjustment of clinical covariates previously identified as risk factors for ascending infection, including oral contraceptive use, gonorrhea co-infection, and chlamydia burden.

Cytokines with adjusted P values < 0.2 by univariable analysis were tested in multivariable parsimonious regression models. The goal of parsimonious regression models is to select as few predictors as possible which best predict an outcome. Here, we determine which subset of cytokines are associated with differential odds of ascending infection. Cytokines were considered significant if P ≤ 0.05. The statistical analyses were performed in R 3.4.2 (The R Foundation for

Statistical Computing).

Relationship between cervical cytokines and reinfection.

To measure the effect of individual cytokines on the risk of reinfection, the hazard ratio

(HR) was determined using a univariable Wei–Lin–Weissfeld Cox model that accounts for repeared chlamydial infections in each person and adjusts for within-subject correlations (Wei LJ

1989). A hazard ratio is a type of survival analysis which measures the effect of an intervention on an outcome. It determines how long it takes for a particular outcome to occur. Previously identified clinical and behavioral risk factors for incident infection (age, gonorrhea infection, chlamydia infection at enrollment, sex with new, uncircumcised, or infected partners) were considered as covariates 116. These covariates, along with chlamydia burden and cytokines with adjusted P values < 0.2 in the univariable model, were selected for stepwise multivariable regression analysis. Cytokines associated with altered risk of reinfection were considered significant if adjusted P ≤ 0.05. The statistical analyses were performed in SAS 9.4 (SAS

Institute Inc.).

33 RESULTS

Baseline characteristics of participants

Cytokine analysis was performed on cervical secretions from 160 chlamydia-infected women; 92 (57%) had cervical infection only, and 68 (43%) had cervical and endometrial infection. Table 2 details the sociodemographic characteristics of study participants. Most were young (median age 20 years; range, 18–35 years), single (91%), African American (66%) women enrolled from ambulatory clinics. The majority (54%) reported having previously received a diagnosis of chlamydia, and 23% reported having ≥ 2 prior infections. Subjects commonly reported past infections with N. gonorrhoeae (22%) and/or T. vaginalis (26%). There were no reports of HIV infection. Of this group, 92 (58%) women completed 4 visits and 126

(79%) completed at least 3 follow-up visits.

Association between cervical cytokines and endometrial infection

The mean and range of values for each cytokine are reported in Table 3. Cytokines involved in neutrophil chemotaxis, survival, and activation (YKL40, CXCL5, GRO, G-CSF, IL-

8, CXCL6) were among the twelve most abundant. We next determined if chlamydia burden affected the level of detectable cytokines. Linear regression analysis revealed that the levels of

38/48 (79%) cytokines in cervical secretions were significantly positively associated with chlamydia cervical burden (Table 4). No cytokine was significantly negatively associated with burden. We showed previously that oral contraceptive use and gonorrhea co-infection were associated with endometrial ascension 116, and Endo+ women had higher chlamydia cervical burdens compared to Endo- women 116. Based on these findings, we treated chlamydia burden, oral contraceptive use, and gonorrhea co-infection as confounders that were adjusted in subsequent analyses. By univariable logistic regression, CXCL10, CXCL9, IL-17A, TNFα, and

34 CXCL13 were associated with increased odds of endometrial infection, while PDGF-AA, IL-15,

CXCL14, PDGF-BB, IL-16, FGF-2, and IL-14 were associated with decreased odds of endometrial infection (adjusted P<0.2) (Table 5). We used P<0.2 as our cutoff for inclusion in the multivariable model. These cytokines were entered into stepwise regression analysis with chlamydia burden, oral contraceptive use, and gonorrhea co-infection to determine the least parsimonious final model for cytokines associated with endometrial infection. CXCL10

(adjusted OR 1.69; 95% CI, 1.09-2.70), TNFα (adjusted OR 1.54; 95% CI, 1.01-2.41), and

CXCL13 (adjusted OR 1.44; 95% CI, 1.03-2.06) were associated with increased odds, while IL-

16 (adjusted OR 0.51; 95% CI, 0.30-0.81) was associated with decreased odds of endometrial infection in the final multivariable model (adjusted P ≤ 0.05) (Table 6). Oral contraceptives and gonorrhea co-infection were not significantly associated with endometrial infection, however cervical chlamydial load was still significantly associated with increased odds of endometrial infection after multivariable analysis.

Association between cervical cytokines and reinfection

We used univariable analysis to determine cytokines associated with reinfection, while adjusting for age, gonorrhea infection, chlamydia infection at enrollment, sex with new, uncircumcised, or infected partners, and chlamydia burden (Table 7). IL-14, CXCL11, CXCL9, and CXCL10 were associated with increased risk, while VEGF, Flt-3L, and TNFSF10 (TRAIL) were associated with decreased risk of reinfection (adjusted P<0.2). IL-14 and VEGF were the most significantly associated (P<0.1). However, no cytokines were significantly associated with reinfection after multivariable analysis that included the above clinical and behavioral covariates.

35 DISCUSSION

Chlamydia trachomatis is a bacterium that causes a wide range of diseases in humans. In women, chlamydia infects single columnar epithelial cells of the endocervix resulting in a modest inflammatory response that is characterized by redness and mucopurulent cervicitis.

Despite infection initiating local inflammation, C. trachomatis infection is asymptomatic in 70-

90% of women 13, leading to women remaining undiagnosed and untreated for extended periods of time. Untreated infections can lead to significant morbidity, including ascension of infection to the upper reproductive. One manifestation of untreated C. trachomatis infection in the upper genital tract is PID. If left untreated, PID can lead to infertility and permanent damage to a woman’s reproductive organs 26. PID can go unrecognized by women and their health care providers when the symptoms are mild, a phenomenon termed subclinical PID. Despite lack of symptoms, histologic evidence of endometritis, which is associated with Fallopian tube infection

(salpingitis), has been demonstrated in women with subclinical PID.

Currently, a major research gap is the lack of a noninvasive procedure that can accurately identify women with subclinical PID. Since chlamydia does not disseminate, the immune profiles associated with disease are likely localized to the site of infection. Chlamydia infection in humans and animal models, upregulates many innate inflammatory mediators within the reproductive tract, and the presence of some of these mediators have been known to be associated with disease. Previous analyses in our lab revealed important human responses that provided a pathway for further biomarker investigation. Endometrial chlamydia infection was associated with elevated type I IFN blood transcripts and increased CXCL10 in cervical secretions 187. To determine if these and additional cytokines were associated with endometrial infection or reinfection, we expanded our analysis by measuring the concentration of 49 cervical

36 cytokines and used a multivariable parsimonious regression model to control for previously identified confounding variables associated with upper genital tract ascension and incident infection 116. We first demonstrated that chlamydia burden was correlated with increased expression of most detectable cytokines. This was unsurprising considering that more bacteria will initiate a stronger inflammatory response and enhanced cytokine production. Based on this finding, we controlled for chlamydia burden, along with additional clinical and behavior variables, in our cytokine analysis. Interestingly, chlamydia burden in the cervix was still significantly associated with increased odds of endometrial infection after multivariable analysis, however oral contraceptive pills and gonorrhea co-infection were not. This suggests that cervical burden may be a critical clinical variable to consider when estimating the odds of endometrial infection and risk of upper genital tract disease.

Additionally, we found that the interferon-induced chemokines CXCL9, CXCL10, and

CXCL11, the proinflammatory cytokines IL-17A and TNFα, and chemokine CXCL13 were also associated with endometrial infection. Interferon-induced chemokines CXCL9, CXCL10, and

CXCL11 are involved in the chemotaxis of T cells. In the mouse model of chlamydia infection,

CXCL9 and CXCL10 were highly expressed in the upper genital tract of mice infected with C. trachomatis 180. Most recently, expression of CXCL9, CXCL10, and CXCL11 were associated with the recruitment of non-protective immune cells in chlamydia genital tract infection and the development of pathology 103. We previously demonstrated that type-1 interferon signaling in mice drives CXCL10 expression, prolonged chlamydia infection, and increased oviduct pathology 102. This is consistent with the type-1 interferon blood transcriptional signature observed in women with endometrial chlamydia infection and endometritis 187.

37 Similarly, proinflammatory cytokines IL-17A and TNFα are associated with upper genital tract pathology in mice. It has been previously demonstrated that during C. muridarum genital infection, IL-17A 92 and TNFα are associated with upper reproductive tract pathology 81.

In IFNγ-deficient mice infected with C. muridarum, there was significantly increased bacterial burden, increased neutrophil recruitment, enhanced production of Th17-differentiating cytokines, predominant Th17 response, increased IL-22 response, and enhanced genital tract tissue damage

93. TNFα has also been shown to cause upper reproductive tract pathology in mice after C. muridarum infection 81 and previous human studies have linked TNFα to infertility in chlamydia- infected women 115,177,188,189. It has also been shown that TNFα producing CD8+ T cells, that are antigen specific, cause upper genital tract pathology in chlamydia infection in mice 81,82. C. trachomatis infected Fallopian tubes express the chemokine CXCL13 and was associated with C. trachomatis infection of the female genital tract 175. Further, preferential expression of CXCL13 in human Th17 cell clones has been reported 190. However, the relationship between cervical

CXCL13 and upper genital tract infection is unclear. CXCL13 is produced by multiple cell types and is a potent recruiter and activator of T follicular helper (Tfh) cells and B cells 191,192.

Neutralization of CXCL13 has been shown to inhibit formation of gastric lymphoid follicles after Helicobacter suis infection 193. CXCL13 may be a marker of increased ectopic lymphocyte cluster development and germinal center activity 194. This would be a potential mechanism for lymphoid aggregation and increased plasma cell frequency observed during chlamydia infection in women with endometritis and salpingitis 195, and may also be related to the high antibody titers observed in susceptible women with repeated chlamydia infection 196-198.

Increased expression of PDGF-AA/BB, FGF, CXCL14, IL-14, IL-15, and IL-16 were associated with decreased risk of endometrial infection. Chlamydia infection induces PDGF and

38 FGF signaling in cells, which may promote wound healing and recruitment of phagocytic immune cells such as neutrophils and macrophages, which can enhance bacterial clearance.

CXCL14 is a chemokine involved in immune surveillance and inflammation 199-201. It is expressed at high levels in normal tissues 202. It is a potent chemoattractant and activator of dendritic cells 203, monocytes 204, neutrophils 205, and NK cells 206. It is stimulated by LPS 202 and has been shown to have direct antimicrobial effects 207,208. CXCL14 may be involved in maintaining tissue resident immune cells. IL-15, an important T cell growth factor, may promote

T cell survival and maintenance. Additionally, IL-15 may promote T cell effector function by upregulating IFNγ production and decreasing IL-17A expression209,210. Finally, IL-16 is a cytokine that is involved in the recruitment of immune cells expressing CD4, such as T cells, dendritic cells, monocytes, and eosinophils. There are no reports of studies examining the role of

IL-16 during chlamydia infection. However, this observation may have important implications.

IL-16 is a potent CD4+ T cell chemoattractant 211 that directly correlates with the number of infiltrating CD4+ T cells in asthmatic epithelium 212,213 and pleural effusions in tuberculosis patients 214. IL-16 can also prime recruited Th1 cells for IL-2 and IL-15 responsiveness 215. Since

Th1 cells are a well-characterized correlate of immune protection against chlamydia 125 127,130-133, cervical IL-16 may reflect infiltration of effector Th1 cells capable of eliminating chlamydia infection at the cervix and preventing endometrial infection.

IL-14 and VEGF were associated with an increased and decreased risk of reinfection by univariable analysis, respectively, but were not maintained in the final multivariable model.

VEGF has been shown to be secreted by T cells during hypoxia and after stimulation by cognate antigen or IL-2 216. Hypoxic conditions also enhanced T cell responsiveness to VEGF by increasing T cell VEGFR2 expression. Furthermore, VEGF enhanced IFN and inhibited IL-10

39 production by T cells during antigen-stimulation, suggesting VEGF can enhance a Th1 phenotype. VEGF has also been shown to function as an amplifier for effector T cell recruitment and activation via Notch signaling 217. IL-14 is a B cell growth factor, produced by B cells and T cells 218. It stimulates B cell proliferation, inhibits antibody production, and expands selected B cell subgroups. The association of IL-14 with reinfection may represent a non-protective humoral response, potentially consistent with CXCL13 expression observed in our analysis of endometrial infection. IL-14-transgenic mice demonstrated increased numbers of B1, B2, and germinal center B cells, along with enhanced antibody responses to T-dependent and T- independent antigens, compared with littermate controls 218.

Limitations of the study include our inability to determine if cytokine responses change with infection duration, how long after infection acquisition the sample was obtained in each participant, or their relationship to potential changes in bacterial burden that might occur over time. Additionally, since cytokine levels were measured in cervical sponge eluates, we were not able to determine their cellular sources. Commercial NAAT tests are highly sensitive but non- quantitative while the qPCR assay used here to determine chlamydial abundance is precise but relatively insensitive because of the small fraction of clinical sample analyzed. Neither assay discriminates infectious chlamydiae from dead and/or degraded bacteria, which have no potential for ascension or transmission. Quantitation of infectious chlamydiae as a reflection of active bacterial replication would be useful to help confirm the association of cervical burden with endometrial infection.

FUTURE DIRECTIONS

One study that evaluated endometrial biopsies from women with suspected pelvic inflammatory disease (PID) found that C. trachomatis infection was associated with an

40 inflammatory milieu consisting of neutrophils, plasma cells, and periglandular lymphoid follicles containing transformed lymphocytes 67. Therefore, cytokines and chemokines that recruit neutrophils, plasma cells, and T cells are likely to be present in women with PID. Indeed, the results of this thesis support this clinical observation. Neutrophils are early responders during chlamydia infection. Chlamydia infected epithelial cells can release TNFα and type I interferons

IFNα and IFNβ, which stimulates the production of CXCL10219. TNFα can activate neutrophils and both type I interferons and CXCL10 are chemoattractants for neutrophils102,220,221. In response to cytokine stimulation, neutrophils can release CXCL10222, which in turn recruits more neutrophils. In the mouse model of chlamydia genital tract infection, neutrophil influx is directly correlated with pathology 61-66. Additionally, CXCL10 is a chemoattractant for T cells. Although

Th1 cells are important for protection against chlamydia, it is likely that the excess of CXCL10 recruits additional T cells that are non-protective. These non-protective T cells may include Th17 cells and CD8+ T cells secreting TNFα. These subsets of T cells are known to contribute to pathology possibly through their recruitment of neutrophils or other non-protective T cells 103.

Additionally, Th17 cells can also secrete CXCL13, a potent chemoattractant for B cells 190.

CXCL13 can expand B cell subgroups, likely plasma cells, which are unable to control the infection 191,192. Further, CXCL13 is highly expressed in and helps to maintain lymphoid organs.

It is possible that CXCL13 is responsible for the formation and maintenance of lymphoid aggregates found in the endometrium of women with PID. Importantly, women with endometrial infection secreted decreased levels of IL-16. IL-16 is a cytokine involved in the recruitment and proliferation of Th1 cells 215. It has been well documented that Th1 cells are critical to bacterial clearance and necessary for protection from reinfection 125 127, 13-133. These data suggest that women with endometrial infection may have trouble recruiting protective Th1 cells to the site of

41 the infection, making them vulnerable to ascending infection. Although other microorganisms are known to cause or contribute to PID 29-34, it is unknown in this cohort whether commensal microorganisms of the vagina or uterus may contribute to risk for endometrial infection. A working model of risk for endometrial chlamydial infection is illustrated in Figure 1.

Future studies can be done to determine the specific cell types that are secreting these cytokine and chemokines. From the TRAC cohort, endometrial and cervical cell samples are available. These samples are from the site of infection and likely contain cell subsets that may contribute to pathology. Using techniques such as single cell transcriptomics or cytometry time of flight (CyTOF) studies can evaluate the cell types present in these tissues and identify the cytokines being produced by the cells. Additionally, research can be done to determine if the increased presence of specific cells is correlated with endometrial chlamydia infection.

Our final multivariable model identified CXCL10, TNFα, and CXCL13 as cytokines most associated with endometrial chlamydia infection while IL-16 was most associated with limitation of infection to the cervix. The final multivariable model led to the development of a predictive equation that can be tested prospectively in a similar cohort of women to predict the presence of endometrial infection. The predicted probability of having endometrial infection can be estimated by the multivariable logistic regression model: Final equation: logit(Pr(Endo+))= -

1.71 +0.52*(CXCL10)+0.43*(TNFa)+0.36*(CXCL13)-0.68*(IL-16) +0.58*(Gonorrhea)-

0.01*(Oral contraceptive pills)+0.72*(Cervical Chlamydial load). Future cohort studies can test how well these cervical cytokines alone can predict endometrial infection in a clinical setting.

Since chlamydia is an infection localized to the genital tract, it is likely that inflammatory markers at the cervix will most reliably predict endometrial infection, however, future studies can also determine whether inclusion of host gene expression signatures in the blood or

42 microbiome information from the vagina or uterus could increase or decrease prediction accuracy, thus improving the equation.

Finally, the mouse model of chlamydia genital tract infection can be used to investigate the specific interactions of these mediators and how the inflammatory responses might be modified therapeutically, to limit tissue damage. For example, increased levels of IL-16 is associated with decreased risk for ascending infection. To date, this is the first report of IL-16 being associated with chlamydia disease risk. Future studies can use IL-16 knockout mice223 to study development of chlamydia-induced pathology. One could hypothesize that the absence of

IL-16 would make chlamydia infected mice more susceptible to ascending infection and reinfection because of a lack of recruitment of protective Th1 cells. Similarly, studies can determine whether administration of exogenous IL-16 is protective against endometrial infection.

Lastly, we show that VEGF is associated with decreased risk for reinfection. Future studies in the mouse model can determine if this association extends to mice and what the mechanism of protection might be. VEGF enhances IFN production and inhibits IL-10 production by T cells during antigen-stimulation, suggesting VEGF can enhance a Th1 phenotype 216. VEGF knockout mice can be used to determine if the Th1 response is reduced in chlamydia-infected mice, thereby increasing susceptible to reinfection.

Chlamydia infection is asymptomatic in most people, which leads to delays in diagnosis and treatment. Untreated chlamydial infections in women can lead to serious consequences including pelvic inflammatory disease (PID), tubal factor infertility, ectopic pregnancy, and chronic pelvic pain. In the clinic, subclinical PID is silent and thus undetected by women, her healthcare provider, and most screening and treatment programs. This work is significant because we have identified cytokine profiles that are associated with subclinical PID, which can

43 be used to assist clinicians with identifying these women in the clinic and ensuring that they are aware of the same risks, treatment opportunities, and counseling that is available to women with clinical PID.

44 Table 1: Univariable regression analysis: Association of cytokines with endometrial chlamydia infection in women with high bacterial burden

Cytokines associated with increased odds of endometrial infection Cytokine Adjusted OR* P Value CXCL10 (IP-10) 2.51 0.01 IL-10 1.83 0.07 CXCL9 (MIG) 1.41 0.25 CXCL6 (GCP2) 1.93 0.41 G-CSF 1.52 0.42 CXCL5 (ENA-78) 1.54 0.44 YKL40/CHI3L1 1.04 0.62 EGF 1.27 0.63 CXCL11 (I-TAC) 1.14 0.64 BCA-1 1.1 0.69 IL-8 1.1 0.71 IL-17A 1.01 0.95 TNFα 0.999 1.00

Cytokines associated with decreased odds of endometrial infection Cytokine Adjusted OR* P Value CXCL14 (BRAK) 0.37 0.01 IL-12p40 0.29 0.01 (CX3CL1) Fractalkine 0.21 0.02 IL-12p70 0.44 0.02 GM-CSF 0.45 0.02 IL-13 0.28 0.02 scd40L 0.36 0.02 Flt-3L 0.27 0.02 IL-7 0.25 0.02 IL-14 0.50 0.04 CCL11(Eotaxin) 0.39 0.06 FGF-2 0.52 0.06 IL-4 0.30 0.06 MCP-3 0.38 0.07 APRIL 0.45 0.08 TGF-α 0.38 0.09 MDC 0.52 0.10

45 VEGF 0.31 0.10 GRO 0.26 0.12 PDGF-AA 0.70 0.13 MCP-1 0.62 0.15 MPIF1 0.62 0.15 RANTES 0.75 0.15 IL-16 0.63 0.18 IFNγ 0.69 0.20 IL-1β 0.70 0.20 IL-23 0.67 0.24 IL-15 0.65 0.28 PDGF-BB 0.79 0.33 IL-1α 0.74 0.33 MIP-1β 0.69 0.41 TNFSF10(TRAIL) 0.78 0.43 IL-1RA 0.70 0.51 MIP-1α 0.73 0.51

* P values adjusted for gonorrhea, Mycoplasma genitalium and oral contraceptive pills (OCPs)

46 Table 2: Sociodemographic characteristics of cohort

Characteristic Value (n=160)

Age, y, median (range) 20 (18-35)

Race

African American 106 (66.3%)

White 31 (19.4%)

Asian 2 (1.2%)

American Indian/Alaska Native 1 (0.6%)

Multiracial 18 (11.3%)

Other race 2 (1.2%)

Ethnicity

Hispanic or Latino 10 (6.3%)

Marital status

Single 146 (91.2%)

Living with partner for ≥4 months 11 (6.9%)

Divorced or separated 3 (1.9%)

Education level

Less than high school graduate 27 (16.9%)

High school graduate or GED degree 65 (40.6%)

Some college work 45 (28.1%)

College graduate 10 (6.3%)

Post-grad work 1 (0.6%)

47 Vocational training 12 (7.5%)

Insurance

None 35 (21.9%)

Private 42 (26.3%)

Public 2 (1.2%)

Don't know 1 (0.6%)

Medicaid 74 (46.2%)

Other insurance 6 (3.8%)

Substance use

Smoking 77 (48.1%)

Marijuana use 63 (39.4%)

Alcohol use 76 (47.5%)

48 Table 3: Descriptive statistics of cytokine levels

Cytokine Mean (SD) Range (pg/ml)

YKL40 (CHI3L1) 32696 (24533) (1713, 147134)

CXCL5 8610 (4193) (1040, 26177)

IL-1RA 7437 (2984) (268, 12951)

GRO 6335 (1976) (767, 11733)

CXCL9 5159 (6873) (68, 41042)

G-CSF 4111 (2399) (311, 13748)

CXCL14 (BRAK) 3147 (2870) (118, 15912)

CXCL10 (IP-10) 2265 (2666) (68, 15433)

IL-14 2076 (1822) (142, 10856)

IL-8 1233 (1626) (101, 16854)

CCL5 (RANTES) 995 (1375) (9, 5680)

CXCL6 933 (306) (194, 1945)

IL-16 740 (691) (63, 4110)

MCP-1 681 (812) (22, 5097)

IL-6 498 (1337) (5, 17752)

FGF-2 399 (500) (49, 3608)

TNFSF13 (APRIL) 288 (188) (40, 1096)

PDGF-BB 282 (543) (13, 4545)

CXCL11 183 (287) (2, 2639)

CX3CL1 (Fractalkine) 147 (75) (14, 459)

IL-1α 124 (310) (3, 4252)

IL-23 118 (107) (13, 781)

VEGF 114 (62) (23, 489)

MDC 88 (94) (17, 1229)

49 TNFSF10 (TRAIL) 85 (91) (5, 919)

CXCL13 (BCA1) 77 (152) (1, 1691)

TGF-α 63 (33) (8, 214)

MIP-1β 57 (52) (5, 386)

PDGF-AA 49 (104) (3, 1189)

CCL3 (MIP-1α) 46 (64) (4, 674)

MCP-3 44 (51) (12, 566)

Flt-3L 38 (21) (7, 177)

IL-12p40 35 (19) (5, 142)

EGF 23 (19) (1, 159)

IL-4 22 (13) (4, 126)

MPIF-1 22 (18) (3, 137)

IL-1β 18 (40) (1, 354)

CCL11 (Eotaxin) 16 (15) (2, 138)

IL-7 14 (5) (5, 30)

TNFα 14 (20) (1, 170)

IL-13 13 (15) (3, 184)

GM-CSF 12 (16) (2, 210)

IL-12p70 12 (6) (3, 50)

sCD40L 12 (9) (1, 70)

IL-17A 10 (10) (1, 73)

IFNγ 9 (13) (1, 162)

IL-10 8 (6) (1, 46)

IL-15 6 (4) (1, 25)

50 Table 4: Association of cytokines with chlamydia burden by univariable regression analysis

Cytokine Correlation coefficient P Value

CXCL13 (BCA1) 0.47 1.35E-09

CXCL9 0.47 1.46E-09

CXCL11 0.43 5.87E-08

CXCL10 (IP-10) 0.43 6.75E-08

IL-17A 0.41 3.07E-07

TNFα 0.39 1.39E-06

MIP-1β 0.36 7.12E-06

IL-16 0.34 2.85E-05

IL-6 0.33 4.31E-05

IL-14 0.32 8.14E-05

MPIF-1 0.31 1.36E-04

TNFSF10 (TRAIL) 0.3 2.15E-04

sCD40L 0.29 3.45E-04

YKL40 (CHI3L1) 0.28 4.88E-04

IL-10 0.28 4.91E-04

TGF-a 0.28 5.29E-04

GRO 0.28 5.92E-04

IL-15 0.28 7.07E-04

IL-12p40 0.28 7.31E-04

CXCL6 0.26 1.32E-03

EGF 0.25 2.08E-03

CCL3 (MIP-1α) 0.25 2.50E-03

CXCL14 (BRAK) 0.25 2.56E-03

51 G-CSF 0.24 3.04E-03

MCP-3 0.24 3.34E-03

CXCL5 0.23 4.53E-03

CCL5 (RANTES) 0.23 5.25E-03

TNFSF13 (APRIL) 0.23 5.61E-03

CX3CL1 (Fractalkine) 0.23 5.74E-03

FGF-2 0.22 6.27E-03

VEGF 0.22 6.60E-03

IFNγ 0.22 6.97E-03

IL-1β 0.22 7.47E-03

PDGF-BB 0.22 8.44E-03

CCL11 (Eotaxin) 0.21 1.09E-02

IL-1RA 0.2 1.75E-02

MDC 0.17 3.43E-02

IL-1α 0.16 4.81E-02

IFNα 0.16 5.28E-02

IL-12p70 0.15 6.29E-02

IL-8 0.15 7.12E-02

MCP-1 0.14 8.42E-02

PDGF-AA 0.14 9.74E-02

GM-CSF 0.13 1.11E-01

Flt-3L 0.11 1.82E-01

IL-23 0.09 2.67E-01

IL-7 -0.05 5.83E-01

IL-13 -0.03 6.96E-01

IL-4 0.01 9.10E-01

52 Table 5: Association of cytokines with chlamydia endometrial infection by univariable regression analysis

Cytokines associated with increased odds of endometrial infection P Adjusted OR* 95% CI Cytokine Value CXCL10 (IP-10) 1.51 (1.11, 2.10) 0.01 CXCL9 1.29 (0.98, 1.73) 0.08 IL-17A 1.36 (0.97, 1.95) 0.08 TNFα 1.29 (0.97, 1.75) 0.09 CXCL13 (BCA1) 1.17 (0.93, 1.50) 0.19 G-CSF 1.36 (0.84, 2.25) 0.22 CXCL11 (I-TAC) 1.17 (0.90, 1.51) 0.23 MIP-1β 1.21 (0.84, 1.78) 0.32 YKL40 (CHI3L1) 1.17 (0.79, 1.75) 0.43 CCL3 (MIP-1α) 1.04 (0.72, 1.48) 0.85 sCD40L 1.01 (0.67, 1.54) 0.95 CXCL5 (ENA-78) 1.01 (0.60, 1.71) 0.96

Cytokines associated with decreased odds of endometrial infection P Adjusted OR* 95% CI Cytokine Value PDGF-AA 0.77 (0.60, 0.96) 0.03 IL-15 0.7 (0.46, 1.04) 0.08 CXCL14 (BRAK) 0.78 (0.58, 1.03) 0.08 PDGF-BB 0.81 (0.63, 1.03) 0.09 IL-16 0.8 (0.58, 1.10) 0.17 FGF-2 0.83 (0.61, 1.09) 0.19 IL-14 0.81 (0.59, 1.10) 0.19 CCL5 (RANTES) 0.89 (0.73, 1.06) 0.20 IL-12p40 0.73 (0.42, 1.25) 0.26 CX3CL1 (Fractalkine) 0.73 (0.42, 1.26) 0.26 IL-4 0.73 (0.41, 1.29) 0.29 GM-CSF 0.81 (0.51, 1.22) 0.33 IL-12p70 0.76 (0.43, 1.32) 0.34 CCL11 (Eotaxin) 0.78 (0.45, 1.30) 0.36 MDC 0.83 (0.54, 1.25) 0.38 TNFSF13 (APRIL) 0.83 (0.53, 1.27) 0.39 EGF 0.84 (0.56, 1.27) 0.41

53 MCP-1 0.9 (0.68, 1.18) 0.46 IFNγ 0.87 (0.60, 1.28) 0.48 IL-6 0.92 (0.72, 1.17) 0.50 Flt-3L 0.84 (0.49, 1.40) 0.50 MCP-3 0.83 (0.46, 1.42) 0.50 IL-1α 0.92 (0.71, 1.19) 0.53 IL-7 0.79 (0.37, 1.66) 0.53 TNFSF10 (TRAIL) 0.9 (0.66, 1.24) 0.53 VEGF 0.86 (0.48, 1.55) 0.63 IL-8 0.93 (0.69, 1.25) 0.64 IL-13 0.9 (0.54, 1.47) 0.67 IL-10 0.9 (0.57, 1.44) 0.67 TGF-α 0.91 (0.56, 1.45) 0.68 IL-1RA 0.95 (0.63, 1.44) 0.82 IL-23 0.97 (0.70, 1.35) 0.86 MPIF-1 0.98 (0.69, 1.39) 0.91 IL-1β 0.99 (0.78, 1.24) 0.92 CXCL6 (GCP2) 0.97 (0.41, 2.32) 0.95 GRO 0.99 (0.47, 2.14) 0.98

* P values adjusted for cervical chlamydia load, gonorrhea, and oral contraceptive pills (OCPs)

54 Table 6: Cytokines associated with chlamydia endometrial infection by multivariable stepwise regression Cytokine OR 95% CI P Value CXCL10 (IP-10) 1.69 (1.09, 2.70) 0.02 TNFα 1.54 (1.01, 2.41) 0.05 CXCL13 (BCA1) 1.44 (1.03, 2.06) 0.04 IL-16 0.51 (0.30, 0.81) 0.01 Gonorrhea* 1.79 (0.52, 6.48) 0.36 Oral contraceptive pills* 0.99 (0.20, 4.90) 0.99 Cervical chlamydial load 2.06 (1.38, 3.23) 7.92E-04 * P values adjusted for cervical chlamydia load, gonorrhea, and oral contraceptive pills (OCPs)

55 Table 7. Association of cytokines with chlamydia reinfection by univariable regression analysis Cytokines associated with increased risk of reinfection Factors Adjusted HR* 95% CI P Value IL-14 1.19 (0.97, 1.45) 0.0929 CXCL11 (I-TAC) 1.14 (0.96, 1.34) 0.138 CXCL9 (MIG) 1.12 (0.96, 1.31) 0.147 CXCL10 (IP-10) 1.12 (0.95, 1.31) 0.174 MCP-3 1.18 (0.83, 1.68) 0.356 CCL11 (Eotaxin) 1.17 (0.83, 1.64) 0.375 CCL5 (RANTES) 1.05 (0.93, 1.19) 0.396 MDC 1.09 (0.84, 1.44) 0.512 CXCL13 (BCA1) 1.04 (0.91, 1.2) 0.54 IL-13 1.12 (0.77, 1.63) 0.542 PDGF-BB 1.04 (0.88, 1.23) 0.625 FGF-2 1.05 (0.85, 1.29) 0.674 IL-10 1.04 (0.83, 1.32) 0.717 IL-17A 1.03 (0.85, 1.24) 0.786 IL-4 1.03 (0.68, 1.57) 0.885 PDGF-AA 1.01 (0.87, 1.16) 0.91 IL-1RA 1 (0.76, 1.33) 0.979

Cytokines associated with decreased risk of reinfection Factors Adjusted HR* 95% CI P Value VEGF 0.71 (0.49, 1.03) 0.0691 Flt-3L 0.8 (0.6, 1.07) 0.128 TNFSF10 (TRAIL) 0.89 (0.76, 1.05) 0.154 IL-8 0.88 (0.71, 1.08) 0.228 IL-7 0.79 (0.52, 1.21) 0.279 MCP-1 0.92 (0.77, 1.09) 0.32 sCD40L 0.88 (0.69, 1.13) 0.328 IFNγ 0.9 (0.73, 1.12) 0.355 TGF-α 0.87 (0.63, 1.19) 0.388 IL-1α 0.93 (0.79, 1.1) 0.4 MIP-1β 0.92 (0.73, 1.15) 0.439 G-CSF 0.88 (0.64, 1.22) 0.457 IL-6 0.94 (0.79, 1.12) 0.464 GM-CSF 0.89 (0.63, 1.27) 0.534

56 IL-23 0.94 (0.75, 1.16) 0.549 IL-1b 0.96 (0.84, 1.11) 0.608 CXCL14 (BRAK) 0.95 (0.78, 1.16) 0.627 MPIF-1 0.95 (0.75, 1.2) 0.654 CXCL6 0.92 (0.56, 1.49) 0.724 YKL40 (CHI3L1) 0.96 (0.77, 1.21) 0.753 IL-15 0.98 (0.77, 1.25) 0.882 IL-12p40 0.98 (0.7, 1.37) 0.907 EGF 0.98 (0.71, 1.36) 0.911 IL-16 0.99 (0.8, 1.23) 0.922 CCL3 (MIP-1a) 0.99 (0.8, 1.23) 0.926 TNFSF13 (APRIL) 0.99 (0.79, 1.25) 0.946 IL-12p70 0.99 (0.68, 1.43) 0.957 GRO 0.99 (0.54, 1.81) 0.968 CX3CL1 (Fractalkine) 0.99 (0.67, 1.48) 0.976 CXCL5 (ENA-78) 0.99 (0.68, 1.47) 0.979 TNFα 1 (0.84, 1.19) 0.98

*Adjusted for cervical chlamydia load, age, gonorrhea, chlamydia infection at enrollment, and sex with new, uncircumcised, or infected partners

Figure 1: Proposed model of endometrial Chlamydia infection

Figure 1: Proposed model of endometrial chlamydia infection. Chlamydia infection of epithelial cells in the endocervix induces production of TNFα and type I interferons. TNFα and interferons synergize to promote the production of CXCL10. Neutrophils, which are early responders in chlamydia infection, are recruited to the site of infection by type I interferons and/or CXCL10. Neutrophils can also secrete CXCL10 which leads to the recruitment of additional neutrophils and T cells. The excess of CXCL10 promotes the recruitment of non-protective T cells such as Th17 cell and CD8+ T cells secreting TNFα. In addition to secreting IL-17A, Th17 cells can secrete CXCL13, which can recruit and expand B cell subgroups such as plasma cells and maintain lymphoid aggregates. Women with endometrial chlamydia infection secrete decreased levels of IL-16, a cytokine involved in the recruitment and proliferation of Th1 cells, which are necessary for protection. The contribution of the vaginal or uterine microbiome to endometrial infection is unknown in this model.

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