CEACAM3: Specific Innate Immunity

Anna Sintsova

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Molecular Genetics University of Toronto

Anna Sintsova CEACAM3: Specific Innate Immunity Doctor of Philosophy Department of Molecular Genetics University of Toronto 2015 Abstract

The bacterial species Neisseria gonorrhoeae remains one of the most prevalent bacterial causes of sexually transmitted infections. Gonorrhea is typically characterized by a painful purulent discharge and inflammation, the outcome of overzealous recruitment of neutrophils, phagocytic immune cells highly specialized in the detection and elimination of microbial pathogens. Although molecular mechanisms of gonococcal pathogenesis have been studied in great detail, the causes for the excessive inflammation and immunopathology that follow infection are still not clear. The human neutrophil-restricted innate immune receptor CEACAM3 has been previously shown to promote opsonin-independent uptake and killing of N. gonorrhoeae by neutrophils. In the course of my thesis work, I have established that the role of CEACAM3 is not restricted to the neutrophils’ direct engulfment and killing of gonococci, since it also drives a vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 represents a tipping point between the protective and pathogenic outcomes of N. gonorrhoeae infection. My biochemical profiling of gonococcal-infected, genetically modified neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ-, Bcl10-MALT1-, and Tak1-dependent signaling cascade that leads to the activation of an NF-κB-dependent transcriptional response, with consequent production of pro- inflammatory cytokines. I also showed that CEACAM cross-linking on neutrophils signals in synergy with TLR4, significantly amplifying the response to LPS, suggesting that CEACAM3 acts as an integral part of a complex innate immune detection network. Finally, I co-led a study that characterized Opa protein variant expression among a collection of clinical specimen-derived N. gonorrhoeae, and then assessed their CEACAM binding profile. This study suggested that mucosal infection in humans selects for binding to epithelial-expressed CEACAMs but against CEACAM3 binding in vivo. Together, my studies implicate a role for CEACAM3 as a host- adaptive innate receptor that promotes gonococcal detection and elimination in the context of natural infection, but also reveal potential pathogenic consequences of CEACAM3 activation.

Acknowledgments

I would like to thank Dr. Scott Gray-Owen for his guidance and his patience. His constant support and encouragement over the last 6 years have made me the scientist I am today. If I learned nothing else, I hope that I will take away with me his enduring optimism and enthusiasm for all things science. I would like to thank my supervisory committee members, Dr. Philpott, Dr. Glogauer, and Dr. Egan, who were always there to keep me on my toes, and whose invaluable ideas greatly helped to shape and guide this project.

I would also like to thank all the members of the Gray-Owen lab, past and present, who are a truly amazing group of people, and who made 6 years seem like a very short time indeed. I want to especially thank Dr. Sarantis, who taught me the ropes of neutrophil work and Epshita Islam who was always there through all the highs and the lows, and kept me sane.

I would like to thank my family for their (sometimes wavering) understanding of why I chose to spend a whole decade of my life at the University of Toronto and their (never wavering) love. I would also like to thank my husband, who has recently taken over Epshita’s responsibility of telling me “We’re going to be all right”, and is doing a stellar job.

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Table of Contents

Table of Contents ...... iv

List of Tables ...... ix

List of Figures ...... x

1 General Introduction ...... 1

1.1 Neisseria gonorrhoeae: clinical manifestation and molecular mechanisms of disease ...... 1

1.1.1 Clinical Manifestation ...... 1

1.1.2 Immune responses to gonococcal infection ...... 2

1.1.3 Neisseria gonorrhoeae pathogenesis ...... 3

1.2 CEACAM family of Receptors ...... 3

1.2.1 CEACAM1 ...... 4

1.2.2 CEACAM5 and CEACAM6 ...... 5

1.2.3 CEACAM3 ...... 5

1.2.4 CEACAMs as microbial receptors ...... 6

1.3 Neutrophil responses to bacterial infection ...... 9

1.3.1 Neutrophil lifecycle ...... 9

1.3.2 Neutrophil-mediated pathogen elimination ...... 10

1.3.3 Neutrophil-derived cytokines and regulation of inflammation ...... 12

1.3.4 Intimate interactions between N. gonorrhoeae and neutrophils ...... 13

1.4 Molecular mechanisms of innate immune response and phagocytosis ...... 14

1.4.1 Fc receptor-mediated phagocytosis ...... 14

1.4.2 Dectin-1 mediated immunity ...... 16

1.5 CEACAM3: specific innate immunity ...... 17

1.5.1 CEACAM3 mediated phagocytosis ...... 17

1.5.2 CEACAM3-driven bacterial elimination ...... 18 iv

1.6 Summary ...... 19

2 Global analysis of neutrophil responses to Neisseria gonorrhoeae reveals a self- propagating inflammatory program ...... 23

2.1 ABSTRACT ...... 24

2.2 INTRODUCTION ...... 25

2.3 MATERIALS AND METHODS ...... 27

2.3.1 Ethics Statement ...... 27

2.3.2 Animals ...... 27

2.3.3 Reagents and Antibodies ...... 27

2.3.4 Bacterial Strains ...... 27

2.3.5 Primary Neutrophil Isolation ...... 28

2.3.6 Human CEACAM Expression in CEABAC Neutrophils ...... 28

2.3.7 Whole Cell Phosphorylation Assays ...... 28

2.3.8 Bacterial infections for immunofluorescence microscopy ...... 28

2.3.9 PMN Killing Assays ...... 29

2.3.10 Oxidative Burst and Degranulation Assays ...... 29

2.3.11 PMN Gene Array Experiments ...... 30

2.3.12 Cytokine Measurements ...... 30

2.3.13 Chemotaxis Assay ...... 30

2.3.14 Air Pouch ...... 31

2.3.15 Neutrophil Depletion ...... 31

2.4 RESULTS ...... 32

2.4.1 CEACAM-humanized transgenic mouse neutrophils respond to N. gonorrhoeae ...... 32

2.4.2 N. gonorrhoeae infection activates CEABAC neutrophils ...... 35

2.4.3 N. gonorrhoeae drives an acute inflammatory program in neutrophils ...... 35

2.4.4 Opa-expressing N. gonorrhoeae drive production of pro-inflammatory cytokines in CEACAM-expressing neutrophils ...... 36

2.4.5 CEACAM-Opa interaction drives an inflammatory response in human peripheral blood neutrophils ...... 38

2.4.6 Phagocytosis and production of reactive oxygen species are not essential for inflammatory cytokine production ...... 38

2.4.7 CEACAM-Opa interaction drives activation of NF-κB and MAPK signaling ..... 42

2.4.8 SFK, Syk, and PKCδ couple CEACAM3 to the downstream transcriptional response ...... 45

2.4.9 CEACAM-Opa intensifies the inflammatory response in vivo ...... 48

2.5 DISCUSSION ...... 53

3 Bcl10 regulates the synergistic inflammatory response elicited by CEACAM3 and TLR4 .... 57

3.1 ABSTRACT ...... 58

3.2 INTRODUCTION ...... 59

3.3 MATERIALS AND METHODS ...... 61

3.3.1 Ethics Statement ...... 61

3.3.2 Animals ...... 61

3.3.3 Reagents and Antibodies ...... 61

3.3.4 Bacterial Strains ...... 61

3.3.5 Primary Neutrophil Isolation ...... 61

3.3.6 Bacterial infections for immunofluorescence microscopy ...... 62

3.3.7 Oxidative Burst Assays ...... 62

3.3.8 Cytokine Measurements ...... 62

3.4 RESULTS ...... 63

3.4.1 CEACAM3 acts in concert with TLR4 to amplify cytokine secretion ...... 63

3.4.2 Rac2-deficient neutrophils readily engulf N. gonorrhoeae, but fail to mount oxidative burst and inflammatory responses ...... 65

3.4.3 Bcl10-deficient neutrophils fail to respond to N. gonorrhoeae infection ...... 69

3.4.4 Malt1-deficient neutrophils mirror the response seen with Bcl10 deficient PMNs ...... 72

3.4.5 Bcl10 and MALT1 deficiency shows selective defect in TLR signaling in CEABAC PMNs ...... 72

3.4.6 Bcl10 regulates the synergistic cytokine production elicited by CEACAM3 and TLR4 ...... 75

3.5 DISCUSSION ...... 77

4 Selection for CEACAM receptor-specific binding phenotype during Neisseria gonorrhoeae infection of the human genital tract ...... 79

4.1 ABSTRACT ...... 80

4.2 INTRODUCTION ...... 81

4.3 MATERIALS AND METHODS ...... 84

4.3.1 Reagents ...... 84

4.3.2 Bacterial strains and growth conditions ...... 84

4.3.3 Cell lines ...... 85

4.3.4 CEACAM receptor expression ...... 86

4.3.5 96-well binding assay ...... 86

4.3.6 Whole Cell Dot Blot ...... 87

4.3.7 Statistical analysis ...... 87

4.3.8 Primary Neutrophil Isolation ...... 87

4.3.9 Bacterial infections for immunofluorescence microscopy ...... 88

4.3.10 Oxidative Burst and Degranulation Assays ...... 88

4.3.11 Cytokine Measurements ...... 89

4.4 RESULTS ...... 90

4.4.1 Most low passage clinical isolates display Opa protein expression ...... 90

4.4.2 Establishing a high-throughput CEACAM binding assay ...... 94

4.4.3 LPCIs show distinct CEACAM-binding patterns ...... 97

4.4.4 CEACAM binding affects human neutrophil responses to LPCIs ...... 99 vii

4.5 DISCUSSION ...... 106

5 General Discussion ...... 108

5.1 CEABAC Mice: ...... 109

5.2 CEACAM-dependent Transcriptional Response: ...... 110

5.3 Specific Innate Immunity: ...... 111

5.4 CEACAM3-dependent Signalling: ...... 112

5.5 CEACAM Interactions: ...... 114

5.6 CEACAMs in Gonococcal Infection: ...... 114

5.7 Neutrophils and the Adaptive Response: ...... 115

5.8 Conclusions: ...... 116

6 References ...... 118

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List of Tables

Table 1-1. CEACAMs as microbial receptors.……………………..……………………………7

Table 4-1. Adhesin expression by a collection of LPCIs..……………………………………..91

List of Figures

Figure 1-1. The CEACAM family and Neisseria gonorrhoeae infection...... ….….…..8

Figure 1-2. Multifaceted role of neutrophils in the immune response..……...…………..….11

Figure 1-3. ITAM Signaling Cascades…...…….……………….…………………….....….20

Figure 2-1. Human CEACAM expression by mouse neutrophils results in neisserial

capture and internalization ….….……………………………………….…..….33

Figure 2-2. N. gonorrhoeae drives an acute inflammatory transcriptional program …...…..37

Figure 2-3. Opa+ N. gonorrhoeae drive CEACAM-dependent production of

pro-inflammatory cytokines in neutrophils …………………….…………...….39

Figure 2-4. Phagocytosis and production of reactive oxygen species are not essential for

inflammatory cytokine production...………………………………………..…..40

Figure 2-5. N. gonorrhoeae infection elicits an NF-κB and p38 MAPK

signaling-dependent cytokine response in CEABAC neutrophil...……….....….43

Figure 2-6. CEACAM3 signaling is required for the PMN cytokine response to

N. gonorrhoeae ……………………………………………...…………....…….46

Figure 2-7. CEACAM binding stimulates the inflammatory response to

N. gonorrhoeae in vivo …………………………………………...…………….49

Figure 2-8. CEACAM3-mediated inflammation promotes immunopathology

associated with N. gonorrhoeae infection …………………………….……….56

Figure 3-1. CEACAM cross-linking enhances LPS-induced cytokine production

by hPMNs ………………………………………………………...…………….64

Figure 3-2. Rac2-deficient CEABAC PMNs readily phagocytose N. gonorrhoeae,

but fail to mount oxidative burst and inflammatory responses ...……………….67

Figure 3-3. Bcl10-deficient neutrophils fail to respond to N. gonorrhoeae infection……….70

Figure 3-4. Contribution of MALT1 to the PMN response to N. gonorrhoeae ……………..73 x

Figure 3-5. Contribution of Bcl10 and MALT1 to the synergistic CEACAM3 and

endotoxin response ……………………………………………….…………….76

Figure 4-1. Opa protein expressing by LPCIs ……………………………………...……….93

Figure 4-2. Establishing 96-well CEACAM-binding Assay.………………………….…….95

Figure 4-3. Low-passage clinical gonococcal isolates show distinct

CEACAM-binding profiles.…………………………………………………….98

Figure 4-4. Male and female isolates show no difference in CEACAM-binding

profiles ………………………………………………………………...………100

Figure 4-5. CEACAM-binding LPCI is readily phagocytosed by human neutrophils.……102

Figure 4-6. Neutrophil bactericidal and inflammatory responses to phenotypic variants

of N. gonorrhoeae ………………………………………………………..……104

Figure 5-1. Schematic representation of CEACAM3 signalling network…………….……113

xi 1

1 General Introduction

The main object of this thesis work is to further our understanding of protective as well as pathogenic neutrophil responses to N. gonorrhoeae infection, with particular emphasis on the role of neutrophil-restricted CEACAM3 receptor. This general introduction will provide relevant background related to N. gonorrhoeae infection and immunity, what is known about The CEACAM family of glycoproteins, especially with regards to their role as bacterial and viral receptors, and a brief overview of our current understanding of neutrophil biology. This is followed by a comprehensive summary of what was known about CEACAM3-mediated responses to N. gonorrhoeae infection prior to the beginning of this work. The aim of this section is to broadly familiarize the reader with this field of research, while more specific and detailed background will be presented in introduction sections for each subsequent chapter. 1.1 Neisseria gonorrhoeae: clinical manifestation and molecular mechanisms of disease

Neisseria gonorrhoeae is a human-restricted bacterial pathogen responsible for the sexually transmitted disease gonorrhea and associated complications, including pelvic inflammatory disease (PID), infertility, and ectopic pregnancies. Despite persistent public health efforts to limit the spread of infection, the level of gonococcal disease are rising [1,2]. Alarming in this regard, the emergence of broad antibiotic resistance, leading to ‘superbug’ strains, and an ongoing lack of success in vaccine development [3,4] have put increased emphasis on research aimed at understanding the immunology and molecular mechanisms of disease.

1.1.1 Clinical Manifestation

Neisseria gonorrhoeae most commonly colonizes the human urogenital tract, although it can be found on other mucosal surfaces as well, including nasopharynx, rectum and conjunctiva. Symptomatic disease is characterized by an overzealous inflammatory response, pain and purulent discharge, and these pathological symptoms are the result of an exaggerated innate immune response rather than overt bacterial-driven cytotoxicity. Disease manifestation varies considerably between men and women. Genital infection in males generally leads to uncomplicated urethritis with prominent neutrophilic discharge. Asymptomatic infections in males are considered infrequent, although estimates vary because it

2 is difficult to accurately evaluate their frequency without widespread sampling [5,6]. In women, N. gonorrhoeae first colonizes and infects columnar epithelial cells of endocervix. Importantly, the rate of asymptomatic infections in women is significantly higher, and is estimated to be between 30 to 80% of all infections depending on the study [1]. Moreover, untreated infections can spread from the endocervix to the upper genital tract, and result in severe sequelae. The infection of the uterus and fallopian tubes can cause scarring, pelvic inflammatory disease, ectopic pregnancies and infertility. The factors contributing to differences in disease manifestations between the sexes are yet to be determined, but may include surface receptor expression, structural, immunological and/or hormonal differences [7]. Potential factors contributing to human disease will be further discussed in chapter 4, where I examine how host’s surface receptor expression might affect the bacterial population.

1.1.2 Immune responses to gonococcal infection

As noted above, gonococcal infection results in a robust innate inflammatory response that is responsible for the immunopathology. While the high frequency of asymptomatic infections would suggest that the inflammatory response is not advantageous to the bacteria, and rather results from its failure to avoid detection, it remains unclear whether the inflammatory response benefits the host or the pathogen. Epithelial cells of the human urogenital tract express a number of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs), which are able to recognize conserved microbial patterns, activate production of pro-inflammatory cytokines, and consequently recruit leukocytes to the site of infection. More specifically, neisserial porin and lipoproteins were shown to activate TLR2 [8,9], while LOS and gonococcal DNA are potent agonists for TLR4 and TLR9, respectively [10,11]. In addition, intracellular receptors NOD1 and NOD2 have been shown to contribute to the innate response against N. gonorrhoeae infection [10]. The initial activation of epithelial cells is a first step in mobilization of host’s innate immune defenses responsible for bacterial elimination. Remarkable when considering the initial severe inflammatory reaction, humans fail to develop a protective immune response to gonococcal infection. Consequently, re-infections with the same strain in previously exposed patients are common [12]. In fact, infection with N. gonorrhoeae elicits a very poor antibody response [13], and does not produce a memory response [14]. Although the exact mechanism for this lack of immunity is unclear, the bacteria

3 have evolved a number of ways to avoid the immune response and suppress the activation of adaptive immunity that will be discussed in more detail below.

1.1.3 Neisseria gonorrhoeae pathogenesis

Extensive in vitro studies, together with insights from the human male urethral challenge studies, have allowed us to establish a model of gonococcal pathogenesis and determine factors essential for this process. An essential first step in N. gonorrhoeae infection is bacterial attachment to the mucosal epithelia, which is mediated by type IV pili. Type IV pili are responsible for initial attachment to the mucosa, and have been reported to bind to CD46 receptor on the cell surface [15], although the receptor identity is still a matter of debate [16]. Following the initial attachment, pilus retraction allows closer association between bacteria and apical surface of the epithelia [17]. The importance of pilus for N. gonorrhoeae pathogenesis is highlighted by the fact that N. gonorrhoeae recovered from males and females with symptomatic infection are uniformly piliated [18,19], and although mutants lacking pilus expression were able to establish infection in male volunteers, the disease symptoms were highly attenuated [20]. An intimate secondary association with epithelial cells and consequent cellular invasion is mediated by Opa adhesins. N. gonorrhoeae encode 11 different opa genes, and expression of each one is phase variable due to the presence of a pentanucleotide repeat sequence within the leader peptide-encoding gene sequence [21-24]. Each Opa protein consists of a β-barrel inserted into the bacterial outer-membrane, and flexible surface-exposed loops that mediate binding to human receptors [25-27]. While a small subset of Opa protein variants mediate attachment to heparan sulphate proteoglycans (HSPGs), the vast majority bind one or more carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) [28,29]. Gonococcal association with epithelial CEACAMs drives bacterial transcytosis through the epithelial layer into the sub-epithelial space [30]. In addition to its role in bacterial mucosal colonization, Opa-CEACAM interaction also plays an important part in immune response to N. gonorrhoeae infection, as described below. 1.2 CEACAM family of Receptors

Chapters 2, 3 and 4 are centered on the interaction of gonococcal Opa proteins and the human family of CEACAM receptors, so a brief overview of CEACAM biology and their role in microbial pathogenesis will help with understanding the multifaceted outcomes of this interaction. CEACAM family glycoproteins belong to the immunoglobulin cell adhesion

4 molecules superfamily (IgCAM). IgCAMs possess at least one immunoglobulin (Ig)-like domain that consists of two β-sheets; the remarkable stability of an Ig-fold and its ability to facilitate interactions with other Ig-like domains explains its widespread utilization across many biological processes [31]. CEACAMs are composed of one IgV-like domain that is necessary to allow homotypic and heterotypic CEACAM-CEACAM interactions, as well as a variable number of IgC-like domains. In humans, the CEACAM family is comprised of 12 members that modulate a wide range of cellular processes, including shaping tissue architecture, neovascularization, regulation of insulin homeostasis, and T-cell proliferation [32]. Interestingly, these proteins have also become targets of multiple bacteria and viruses that use them to establish infection and colonization. Here I focus on four members of the family that actively or passively participate in host-pathogen interaction: CEACAM1, CEACAM3 CEACAM5 and CEACAM6 [32,33].

1.2.1 CEACAM1

CEACAM1 is expressed by a wide range of tissues, and can be found on epithelial cells, endothelial cells, leukocytes, and myeloid cells [34-37]. The outer-membrane portion of CEACAM1 consists of one IgV-like, and up to three IgC domains, while the cytoplasmic domain of the most abundant (long) isoform contains two functional immunoreceptor tyrosine- based inhibitory (ITIM) motifs that generally function to inhibit or repress intracellular signaling pathways [38]. A CEACAM1 homologue is found in all vertebrates, including mice and rats, which are used as in vivo models to study CEACAM1’s functions [39]. Thus, it was originally observed that CEACAM1 is involved in neovascularization and angiogenesis, as CEACAM1 deficient mice have impaired vascular remodeling after ischemia [40-42]. CEACAM1 also plays a role in regulation of insulin function, as mice expressing a dominant negative form of CEACAM1 show impaired clearance of insulin in the liver [43]. In accordance with CEACAM1 having a general function in control of cell growth and proliferation, CEACAM1-deficient animals show increased tumor formation after induction with azoxymethane [44]. This growth and activation suppressive function is also evident in control of the immune response, where extensive studies have established an important role for CEACAM1 in inhibition of T cell responses [37,45-49], monocyte survival [50], normal maturation of dendritic cells required to elicit an adaptive immune response [51], and in NK cell-mediated killing of transformed cells [52]. More recently, CEACAM1 has also been shown to regulate granulopoiesis, as CEACAM1-deficient mice have neutrophilia, which makes them more susceptible to Listeria

5 monocytogenes infection [53]. CEACAM1 has also been shown to down-regulate TLR4- induced IL-1β secretion in neutrophils [54] and to limit neutrophil-driven blood-brain barrier damage after stroke [55,56].

1.2.2 CEACAM5 and CEACAM6

Epithelial-expressed CEACAM5 (also known as CEA) and CEACAM6 are composed of one IgV-like domain with six and two IgC domains, respectively. Unlike CEACAM1, CEACAM5 and CEACAM6 lack a cytoplasmic domain since they are anchored in the membrane via a glycosylphosphatidylinositol (GPI) linkage [32,57-61]. CEACAM5 and CEACAM6 are found only in primates; no homologues are found in mouse genome [62]. CEACAM5 expression is limited to epithelium, with the highest levels found on gastrointestinal cells [34]. In healthy individuals it can also be found on epithelial cells of the tongue, esophagus, stomach, sweat glands, prostate [34,63] and vagina [Islam et al, manuscript in preparation]. In malignant transformation, CEACAM5 is aberrantly upregulated in the lung, pancreas, gallbladder, urinary bladder, and in the female genital tract [34]. CEACAM6 has a broader expression pattern, and in addition to being found on many epithelial cells, it is expressed on granulocytes [64]. CEACAM5 and CEACAM6 have mostly been studied in the context of cancer. Due to its abnormal appearance on many transformed cells of the large intestine, CEACAM5 has long been used as a marker for colorectal tumors [65]. CEACAM6 is also overexpressed in a variety of cancers, and predicts poor survival [66]. CEACAM5 functions in cell-to-cell adhesion [67], where homophilic binding inhibits cellular differentiation and prevents apoptosis [68,69]. Overexpression of either CEACAM5 or CEACAM6 leads to disturbance of ordered tissue architecture in 3D cultures of colon carcinoma cell lines [70], increases susceptibility to colon cancer in transgenic mouse model after azoxymethane treatment [71], and inhibits apoptosis [69]. Although signaling downstream of GPI-anchored CEACAMs is not well understood, at least in case of CEACAM5 it seems to involve activation of integrin signaling in lipid rafts [72].

1.2.3 CEACAM3

CEACAM3 is a unique member of the CEACAM family, as it is exclusively expressed on human granulocytes and does not interact with other CEACAMs. Indeed it functions to mediate bacterial recognition and elimination by the host’s immune system. Its biological

6 structure and functions are addressed in more detail below in the context of other immune receptors (Section 1.5).

1.2.4 CEACAMs as microbial receptors

The broad mucosal tissue distribution of CEACAMs and their importance for maintenance of a wide range of biological processes make this family of glycoproteins a perfect candidate to serve as receptors for invading microorganisms. A number of murine and human- specific pathogens have independently evolved adhesins that allow binding to various members of CEACAM family to facilitate colonization and invasion of epithelia (Table 1-1). Murine hepatitis virus uses CEACAM1 as a receptor, and CEACAM1-deficient mice are completely resistant to infection [39,73]. In humans, certain strains of Escherichia coli and Salmonella recognize sugars on glycosylated CEACAM1, CEACAM5 and CEACAM6 [74-76]. Moreover, pathogenic and commensal members of Neisseria, as well as Moraxella catarrhalis and Haemophilus influenzae, each express specific proteins that are able to bind to one or more CEACAMs and mediate tight association between the bacteria and epithelia. In an intriguing case of convergent evolution, this diverse group of bacteria uses structurally distinct adhesins to bind epithelial CEACAMs. While Salmonella species and E.coli use fimbrial adhesins for CEACAM attachment [33,77], autotransporter UspA1 [78] and outer membrane protein (Omp) P5[79] (or P1 [80]) serve the same function in M. catarrhalis and H. influenzae, respectively. As mentioned above, Neisseria sp. instead express Opa proteins that exhibit remarkable specificity and ability to differentiate between different CEACAMs [81]. Expression of CEACAM1, CEACAM3, CEACAM5 and/or CEACAM6 is enough to allow internalization of CEACAM-binding bacteria by a variety of different phagocytic and non-phagocytic cell lines [78,79,81-88]. Moreover, transwell cell culture models were used to show that CEACAMs can facilitate transcytosis of bacteria through intact epithelia, allowing them to access sub-epithelial space, a nutrient-rich environment free of other bacterial competitors [30]. Binding to CEACAM1 also allows bacteria to take advantage of the immunosuppressive effect of CEACAM1, since engagement of this receptor can inhibit T cell activation, dendritic cell (DC) differentiation, and signaling through TLR2 [45,49,51,89-93]. These effects can potentially contribute to the fact that N. gonorrhoeae does not generate a protective adaptive immune response. The role of CEACAM-Opa interactions in N. gonorrhoeae pathogenesis is summarized in Figure 1-1.

Table 1-1. CEACAMs as microbial receptors. The biological function and tissue distribution of CEACAMs targeted by bacterial pathogens.

Figure 1-1. The CEACAM family and Neisseria gonorrhoeae infection. N. gonorrhoeae colonization of the human genital tract is facilitated by Opa-CEACAM interaction. a. Opa- CEACAM interaction allows tight association between bacteria and epithelial cells, and transcytosis to subepithelial space. b. Opa-CEACAM1 interaction allows N. gonorrhoeae to suppress and evade the host’s adaptive immune response. c. Presence of the innate-immune decoy receptor CEACAM3 in humans allows efficient opsonin-independent phagocytosis and elimination of N. gonorrhoeae

9 1.3 Neutrophil responses to bacterial infection

This thesis work closely examines the biological function of CEACAM3, a human neutrophil-restricted receptor. As a result of its restricted expression, CEACAM3’s functional responses are closely linked with the biology of a neutrophil. This section is designed to introduce the traditional view of neutrophil, as well as new and emerging concepts about the role of these cells in the innate immune response. Neutrophils, also known as polymorphonuclear leukocytes (PMNs), are an essential part of the innate immune response, and are the first cells recruited to the site of infection. PMNs were originally thought of as short-lived ‘professional killers’ that quickly respond to infection by unleashing a pre-stored arsenal of antimicrobials, after which they are quickly removed by macrophages to limit bystander damage to surrounding tissue. However, recent studies have uncovered new roles for neutrophils beyond the acute inflammatory response, with a plasticity in functional phenotypes [94] that have different roles in microbial clearance, chronic inflammation [95,96], inflammation resolution [97], shaping of adaptive immune response [98,99], as well as tumor immunity [100].

1.3.1 Neutrophil lifecycle

Neutrophils are the most abundant circulating leukocyte in humans (50-70% of white blood cells). However, in mouse blood, PMNs constitute a somewhat lower proportion of circulating white blood cells (10-25%), and there is a greater relative abundance of lymphocytes [96,101]. Neutrophils are continuously generated from common myeloid precursor cells in the bone marrow and are released into the bloodstream once fully differentiated. While a large number of cells remain in circulation, marginalized granulocyte populations can be found in the bone marrow, spleen, liver and lung [102]. Although early work suggested that mature neutrophil’s half-life not to exceed 8 hours in humans [102], this was brought into question by a recent study suggesting that this time can exceed 5 days [103]. During homeostatic conditions, senescent neutrophils are eventually removed by resident macrophages in bone marrow and liver [102]. During infection, a neutrophil recruitment cascade is initiated by epithelial cells and by tissue resident leukocytes that secret inflammatory mediators on contact with microbes. This, in turn, results in an up-regulation of adhesion molecules by the endothelia, which allows tethering of circulating PMNs to initiate slow rolling, adhesion and finally transmigration of the neutrophil from the bloodstream into infected tissue (Reviewed in [96]).

10 1.3.2 Neutrophil-mediated pathogen elimination

Neutrophils are professional phagocytes and express a variety of immunoglobulin Fc domain-specific receptors and complement receptors, as well as C-type lectin receptors, which together allow efficient phagocytosis and destruction of both opsonized and non-opsonized pathogens [104]. Upon microbial uptake, neutrophils deploy a variety of strategies aimed at pathogen destruction. These can be broadly divided into three general categories: 1) release of toxic proteins stored in cytoplasmic granules, known as degranulation, 2) oxidative burst, and 3) release of neutrophil extracellular traps (NETs) (Fig. 1-2). These processes are described below.

1.3.2.1 Degranulation responses Mature neutrophils carry three distinct sets of granules that are formed consecutively during cell differentiation in the bone marrow. Primary (azurophilic) granules are the first to be formed and contain a number of serine proteases (neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G), as well as myeloperoxidase enzyme (MPO), antimicrobial cationic peptides known as defensins, and lysozyme – each of these are highly toxic to bacteria, but can also be responsible for host tissue damage. Secondary (specific) granules are characterized by the presence of lactoferrin protein, and carry a number of other antimicrobial compounds, including neutrophil gelatinase-associated lipocalin [10], human cathelicidin, hCAP-18, that is processed into antimicrobial peptide LL-37, and lysozyme [95]. Tertiary (gelatinase) granules carry few antimicrobials, but are loaded with a number of metalloproteinases (such as gelatinase), while potentially facilitate PMN transmigration. Neutrophils also carry secretory vesicles that contain integrins and other membrane proteins that are important for neutrophil adhesion to the endothelia and migration towards infected sites [95,96,105]. Interestingly, CEACAMs are also found in neutrophil granules, where CEACAM6 is localized to primary granules, while CEACAM1 and CEACAM8 are within specific granules [106].

1.3.2.2 Oxidative burst Oxidative burst results in production of reactive oxygen and nitrogen species (ROS and RNS, respectively). This process is initiated via assembly of NADPH oxidase on the phagosome membrane following bacterial engulfment. NADPH oxidase reduces molecular oxygen to - superoxide (O2 ). Superoxide can then be converted to a number of different intermediates, such

Figure 1-2. Multifaceted role of neutrophils in the immune response. a. Neutrophils secret a wide range of chemokines and cytokines that play a role in leukocyte trafficking, immunomodulation and aid in shaping the immune response. b. Neutrophils are very efficient phagocytes that rapidly degrade ingested microbes via oxidative burst and degranulation responses. c. Under certain conditions, neutrophils can release chromatin structures coated in histones and antimicrobials, known as neutrophil extracellular traps (NETs), which allow them to trap and kill extracellular bacteria.

12 as peroxide (H202) or hydroxyl radicals (OH). MPO can further convert peroxide into hypochlorous acid (HOCl) and chloramines [107]. Besides the direct antimicrobial activity of ROS, these molecules also play an important role as mediators of cell signaling. Consequently, genetic defects causing ablation of ROS production by neutrophils leaves patients vulnerable to a number of infections [95,108].

1.3.2.3 Neutrophil extracellular traps (NETs) In response to a variety of inflammatory stimuli, neutrophils release nucleus-derived structures composed of chromatin DNA, histones and granular proteins, known as NETs. These structures have been shown to be able to trap and limit the spread of extracellular bacteria, and have direct anti-microbial activity against a variety of bacterial pathogens [109-114]. Beyond that, NET formation has also been implicated in vascular obstruction during sepsis [96,112], so this dramatic response may have both protective and pathological effects.

1.3.3 Neutrophil-derived cytokines and regulation of inflammation

The long held view of a neutrophil as a short-lived cell highly specialized for microbial killing is slowly being abandoned, leading to re-evaluation of neutrophils’ role in regulation and orchestration of both acute inflammation and adaptive immune response. Cytokines represent an integral component of signaling networks that allow various cells of the immune system to respond in cohesive and organized fashion to a microbial or other threat. Cytokines constitute a large family of small proteins that can be produced by both immune and non-immune cells with diverse but often overlapping functions, which includes leukocyte recruitment, cell differentiation, activation of B and T cell responses, angiogenesis, and wound repair [115,116]. Studies with human peripheral blood PMNs (hPMNs), as well as mouse studies (both in vitro and in vitro) have shown that PMNs can secret a wide range of cytokines. Although in vitro studies suggest that neutrophils generally produce fewer cytokines per cell than do mononuclear cells, in infected tissues the neutrophils often outnumber other cell types by a few orders of magnitude, making their contribution to cytokine production of considerable importance [98,116-118].

1.3.3.1 Chemokine production The main function of chemokines is to regulate leukocyte trafficking, and secretion of distinct chemokines allows recruitment of discrete cell populations to site of infection. Neutrophils have been shown to secrete a number of chemokines, including IL-8, Groα, IP-10,

13 MIP-1α, MIP-1β, and MIP-2 [98,117]. The importance of these and other neutrophil-derived chemokines for recruitment of T cells [119], including Th17 [120] and Tregs [121], as well as DCs [122,123] and macrophages [124], have been shown in a variety of mouse models. Moreover, considering some of these chemokines are chemotactic for neutrophils themselves, it seems reasonable that PMNs can play a role in amplifying their own numbers at the site of infection [125].

1.3.3.2 Immunoregulatory cytokines In addition to chemokine secretion, PMNs have been shown to be an important source of a number of immunoregulatory cytokines, specifically IL-17 and IFNγ, which are important for host defense against a variety of bacterial pathogens [116,126-130]. Furthermore, neutrophil- derived members of TNF cytokine family (BAFF and APRIL) are important for activation of innate-like B cells [131-133].

1.3.4 Intimate interactions between N. gonorrhoeae and neutrophils

As mentioned above, an overzealous neutrophil response is a hallmark of a symptomatic gonococcal infection. Consequently, a large body of work has been dedicated to understanding the complex relationship between gonococci and this phagocytic cell type. Considering the high level of adaptation exhibited by N. gonorrhoeae to ensure survival within its preferred niche, it comes as no surprise that these bacteria have evolved a number of strategies that allow them to elude a classical phagocytic pathway and consequently evade neutrophil killing [134]. It appears that neutrophils cannot effectively eliminate extracellular Neisseria gonorrhoeae, as inhibition of phagocytosis limits bacterial killing [135,136]. Thus phagocytosis is an essential step in the PMN killing response, and while molecular mechanisms of the process are described below, it is important to understand that traditionally efficient bacterial phagocytosis is viewed as being driven by opsonin receptors (i.e. complement and Fc receptors) and requires a deposition of either complement or antibody on the surface of the bacteria. Only more recently has it become clear that a wider range of phagocytic receptors can recognize pathogens directly. However, classical phagocytosis is clearly an important part of the immune response to N. gonorrhoeae, as gonococci have evolved a number of strategies to avoid antibody and complement binding [137-141]. In addition to avoiding phagocytosis, gonococci can also resist neutrophils’ antimicrobial responses; N. gonorrhoeae expresses a number of enzymes that allow it to detoxify or suppress the oxidative burst response [136,142-148], which may explain

14 why non-oxidative killing is a major contributing factor mediating gonococcal control [134,149]. The close association between the bacteria and its host does not only drive evolution of immune resistance mechanisms in the bacteria, but also puts a selective pressure on the host to come up with novel strategies to combat the invader. Thus, we and others propose that evolutionarily new CEACAM3 is an important host adaptation that allows opsonin-independent phagocytosis and activation of the neutrophils’ antimicrobial responses. This will be further discussed in Chapters 4 and 5. 1.4 Molecular mechanisms of innate immune response and phagocytosis

This thesis work provides an in depth investigation of CEACAM3 biology, which is largely defined by activation of its immunoreceptor tyrosine-based activation (ITAM) motif located in the receptor’s cytoplasmic tail. ITAM motifs are found on a range of immune receptors, and in the context of an innate immune response are important triggers of phagocytosis as well as antimicrobial responses, such as degranulation or activation of inflammatory cytokine production. Innate immune responses rely on the ability of professional phagocytic cells, such as macrophages, neutrophils and dendritic cells (DCs), to take up and eliminate potentially dangerous particles, be they microbes, apoptotic cells, or cell debris. This section focuses on innate immune responses triggered by Fc receptors, which have long been a paradigm for opsonin-dependent particle uptake, and by the C-type lectin receptor Dectin-1 that can directly bind to its target [150]. The signals induced by these receptors has been used to guide our efforts to define a CEACAM3 signaling network and are therefore discussed in detail below.

1.4.1 Fc receptor-mediated phagocytosis

Fc receptors represent a large family of cell surface proteins expressed on myeloid cells that are able to bind the Fc portion of immunoglobulins (Igs), resulting in activation of a number of cellular processes. In general, Fc receptors can be divided into activating (ITAM associated) and inhibiting (ITIM associated) types depending on signaling motif encoded in their cytoplasmic tail, as well as on their affinity for various classes and subclasses of monomeric Igs [134,150-153]. Cell types, and cell subpopulations vary in their expression of specific Fc receptors.

15 The phagocytosis of IgG-coated particles via FcγRIIA, a single chain receptor containing an ITAM motif in its cytoplasmic tail, has become the paradigm for all phagocytic signaling, with the process being subdivided into target binding, signal initiation, and internalization of particles. Although originally thought of as a passive process, the initial target binding is mediated by actin-rich membrane protrusions that are produced by phagocytes to constantly probe the environment for foreign particles [154,155]. Target binding is closely followed by receptor oligomerization, which allows for target immobilization, as well as initiation of phagocytic signaling. Signal initiation is a tightly regulated process that requires effective receptor clustering [156], and is conveyed via ITAM motifs located in the receptor cytoplasmic tail or through an associated adaptor chain. ITAMs consist of two YXXL/I motifs separated by 6-12 amino acids, where X represents any amino acid. Fc receptor engagement and clustering leads to activation of plasma membrane tethered Src family protein tyrosine kinases (PTKs), which phosphorylate tyrosines in the ITAM sequence. This, in turn, recruits and activates Syk tyrosine kinase by virtue of its paired phospho-ITAM-specific SH2 domains docking to the dually phosphorylated FcR cytoplasmic tail. Syk activation is essential for initiation of downstream signaling events; it can employ a number of adapter proteins (i.e. SLP- 76, Gab2, LAT, Nck) and function as an adapter itself, through directly binding and activation of phosphatidylinositol-3-kinase (PI3K) and guanine exchange factor Vav. A primary outcome of these events is recruitment and activation of Rho family GTPases (Rac, Cdc42 and RhoA) that are responsible for actin cytoskeleton-driven formation of a phagocytic cup followed by particle internalization [150-152,157] (Fig. 1-3A). Phagocytosed particles are targeted for degradation, although the exact route might vary depending on the specific phagocytic receptor [158-160]. In addition to its direct function in elimination of microorganisms, Fc receptor engagement has important inflammatory, anti-inflammatory, and/or and immuno-modulatory effects (depending on specific receptor involved) that help shape the innate and adaptive immune response to infection [161]. Cross-linking of activating Fc receptors on macrophages can induce production of pro-inflammatory cytokines, and decrease susceptibility to infection [162,163]. Similarly, activating Fc receptors expressed by DCs can induce production of pro- inflammatory cytokines that support Th1 responses, and enhance antigen presentation [164,165]. In other models, however, signaling via Fc receptors seems to have an anti-inflammatory effect, so the signaling outcomes appear to be context dependent. Presumably because it unleashes such potent responses, ITAM-mediated activation must be tightly regulated to avoid development of

16 autoimmunity as well as immune-mediated damage to the host, and is often counter-balanced by ITIM-mediated suppression of intracellular signaling [160,166-168]. In neutrophils, Fc receptors have been shown to activate cytotoxic responses, however little is known about ITAM-mediated induction of inflammation [104].

1.4.2 Dectin-1 mediated immunity

Dectin-1, a C-type lectin receptor (CLR), which detects beta-glucans in the fungal cell wall, is the best-characterized opsonin-independent phagocytic receptor. Dectin-1 is highly expressed on myeloid cells, and has been shown to be of central importance for host response to fungal infection [169-173]. Phagocytosis is initiated via an ITAM-like motif in the Dectin-1 cytoplasmic tail, and this process shares many similarities with Fcγ receptor-mediated phagocytosis. Dectin-1 crosslinking triggers activation of Src PTKs, as well as Syk, Vav, and Rac, although how essential each of these are for phagocytosis is still not clear, and might be cell type dependent [150,174-178]. For example, uptake of unopsonized zymosan is dependent on Syk activation in bone marrow derived DC, but not in the RAW264.7 macrophage cell line or in IFNγ-induced bone marrow macrophages. In addition to triggering phagocytosis, Dectin-1 was shown to activate a robust oxidative burst in mouse macrophages, neutrophils and DCs [179]. Importantly, Dectin-1 was the first pattern recognition receptor outside of the TLR family that linked recognition of extracellular pathogens to gene transcription, and there has been a great deal of interest in defining the intracellular signaling pathways leading up to these events. The key signaling axis downstream of Dectin-1, which is now known to be common for all ‘activating’ CLRs, involves phosphorylation of the cytoplasmic ITAM-like motif, recruitment and activation of Syk, with subsequent activation of PKCδ, the CARD9-Bcl10- MALT1 (CBM) signaling complex, and NF-κB transcription factors [176,180-182]. However, the details of this response may also be cell type specific, as Dectin-1-CARD9 signals fail to induce NF-κB activation in bone marrow derived macrophages [182]. Other studies have shown that Dectin-1 activates NFAT, IRF1, and IRF5 transcriptional factors, as well as the noncanonical NF-κB pathway [183-186]. Thus, Dectin-1 engagement results in production of pro- inflammatory cytokines and chemokines, including cytokines involved in polarization of adaptive response towards Th17 immunity, which is essential for mucosal immune defense against fungal pathogens [186-189]. Dectin-1 is also able to protect T/B cell deficient mice from re-infection with C. albicans by epigenetically reprogramming monocytes, as pre-exposure of

17 monocytes to β-glucans led to stable changes in histone trimethylation at H3K4 on innate immune genes, which correlated with enhanced transcriptional response upon re-exposure. [190,191]. Although originally studied only in the context of fungal immunity, Dectin-1 was shown to facilitate defense against Mycobacterium tuberculosis [192]. The discovery and characterization of Dectin-1 and other CLRs as sensors of extracellular pathogens has revealed that our innate immune response does not rely on TLRs alone, and is equipped with a much broader range of PRRs than originally imagined [170,172]. 1.5 CEACAM3: specific innate immunity

While most CEACAM family members are, by definition, cell-cell adhesion molecules, CEACAM3 does not seem to bind any other human cell surface receptor. Tellingly, the cytoplasmic domain of CEACAM3 contains an ITAM sequence, implicating a function in immunity [193]. CEACAM3 is a human-restricted member of the CEACAM family that is exclusively expressed on neutrophils. Since it became apparent that CEACAMs were targeted by microbial adhesins [81,88], it has become clear that CEACAM3 serves to facilitate bacterial recognition and elimination by the host’s immune system [194-197]. CEACAM3 shares high sequence similarity with the N-terminal domain of CEACAM1 and CEACAM6, which is the site for bacterial binding; this allows it to bind to neisserial Opa adhesins [198,199]. However, while it is apparent that binding to epithelial CEACAMs is advantageous to the bacteria [30,200,201], CEACAM3-Opa interaction results in enhanced bacterial phagocytosis and killing by neutrophils [196]. Consequently, CEACAM3 has been characterized as a ‘decoy’ innate immune receptor specific for CEACAM-binding pathogens. CEACAM3 has been shown to bind and phagocytose a range of human specific pathogens, including N. gonorrhoeae, N. meningitidis, H. influenzae, and M. catarrhalis [196]. The molecular mechanisms of CEACAM3 signaling have been studied almost exclusively in the context of N. gonorrhoeae infection.

1.5.1 CEACAM3 mediated phagocytosis

Despite its high degree of homology to epithelial CEACAMs, the CEACAM3 domain structure highlights important distinctions. CEACAM3 lacks any IgC domains, and is simply composed of an extracellular IgV-like domain, a single-pass transmembrane sequence and a cytoplasmic tail containing an ITAM-like sequence [194,202]. As a consequence of this, CEACAM3-mediated phagocytosis is strikingly different from uptake mediated by epithelial

18 CEACAMs, and instead bears close resemblance to traditional phagocytosis mediated via ITAM-associated Fc receptors. CEACAM3 interaction with gonococcal Opa adhesins results in tyrosine phosphorylation of its ITAM-like motif via Src PTKs, as CEACAM3-dependent phagocytosis is inhibited by Src-specific inhibitors; and SH2 domains of Src PTKs Hck and Fgr were shown to associate with CEACAM3 in vitro [203-205]. In addition to CEACAM3, human PMNs express high levels of CEACAM1 and CEACAM6, however uptake through these receptors seems to be independent of Src PTKs; while CEACAM1- and CEACAM6-mediated uptake relies on their localization to lipid rafts, the mechanism by which they facilitate engulfment of bacteria remains unexplained [203,205,206]. Since CEACAM-mediated phagocytosis of N. gonorrhoeae by human PMNs is insensitive to cholesterol depletion, CEACAM3 appears to be the major route of uptake of unopsonized Opa-expressing (Opa+) gonococci by human PMNs [194]. By analogy to Fc receptor phagocytosis, CEACAM3-driven bacterial uptake involves formation of actin-rich phagocytic cups, which correlates with GTP loading of Rac GTPase [88,196,203,207]. Dominant negative Rac, but not dominant negative Cdc42, interfered with bacterial uptake into human PMNs [196,208]. Rac activation could be facilitated via Syk- dependent pathways, as CEACAM3 phosphorylation results in Syk kinase recruitment and phosphorylation. However, unlike Fcγ receptors, CEACAM3 was shown to internalize bacteria even in the presence of Syk specific inhibitors, as well as in Syk deficient cell lines [195,208], suggesting a possibility of alternative pathway. This could be partially explained by the fact a Syk downstream effector, Vav, which is responsible for activation of Rho GTPases, was shown to bind CEACAM3 ITAM directly, bypassing requirement for Syk [197] (Fig. 1-3B). However, although bacterial uptake and uptake of small beads coated with CEACAM-specific antibody was not affected by Syk inhibition, uptake of larger particles was significantly diminished. Since CEACAM3 is not naturally expressed in cells that lack Syk, it is unclear whether this Syk- independent pathway would ever be engaged. Regardless, considering that Opa proteins are known to cause bacterial aggregation and formation of ‘microcolonies’, CEACAM3’s Syk- dependent ability to engulf large particles presumably has important implications for infection [195].

1.5.2 CEACAM3-driven bacterial elimination

Following gonococcal uptake by CEACAM3, the bacteria are rapidly killed via activation of neutrophil antimicrobial responses. Several studies have shown that Opa+ N.

19 gonorrhoeae are killed more rapidly by human PMNs than are bacteria lacking Opa expression [196,209,210]. However, because hPMNs express multiple CEACAMs on their surface, it has been challenging to directly attribute bacterial killing to a specific receptor. Although some antibody-based in vitro studies suggested that all three CEACAMs could activate neutrophils antimicrobial responses [211], this does not seem to hold true in the context of N. gonorrhoeae infection. A study using mouse promyelocytic cell line (MPRO) differentiated into functional neutrophils showed that antimicrobial responses are directly dependent on CEACAM3. MPRO cells transfected with CEACAM1, CEACAM3 or CEACAM6 were each able to internalize N. gonorrhoeae, however actin-based phagocytic cups only occurred when CEACAM3 was engaged. Moreover, oxidative burst and degranulation responses were only apparent when CEACAM3-expressing cells were infected with Opa+ N. gonorrhoeae [212]. The CEACAM3- driven ROS production was dependent on Syk kinase, PI3K, and rapid translocation of NADPH oxidase components to gonococci-containing phagosomes [195,213,214]. Notably, while oxidative burst is a clear indication of a robust neutrophil response, several studies report that it does not effectively kill gonococci [134,135,142-145,147,215]. However, CEACAM3 engagement also leads to release of primary and secondary granule contents into phagolysosomes, and treatment with Src PTK or Syk inhibitors prevents degranulation, thereby improving bacterial survival [195,210,212]. Despite the mounting evidence that CEACAM3 is a key receptor involved in rapid recognition and elimination of N. gonorrhoeae, and potentially other CEACAM-binding pathogens, to date little is known about the role of CEACAM3 during in vivo infection. One study showed that Opa proteins expressed by N. gonorrhoeae isolated from a patient with disseminated gonococcal infection failed to bind to CEACAM3, hinting that there was selection against CEACAM3 binding during invasive manifestations of disease [216]. Despite this, all gonococcal strains seem to encode CEACAM3-binding Opa alleles, so their contribution to in vivo infection and disease warrants further investigation. 1.6 Summary

Genitally-derived purulent specimens containing neutrophils associated with Gram negative diplococci have been the defining feature of gonorrhea for generations, yet whether this represents an effective host response or an effective gonococcal virulence strategy has remained

21 Figure 1-3. ITAM Signaling Cascades. A. General mechanism of ITAM-mediated signaling. The general scheme of signal transduction through ITAMs, as shown for Fc and Dectin-1 receptors. Signal transduction is initiated through phosphorylation of ITAM tyrosines by Src PTKs. This results in recruitment and activation of Syk kinase, which conveys the signal to a variety of downstream effectors. The exact proteins involved vary between contexts and cell types. ITAM-signaling results in phagocytosis and oxidative burst response, which depend on Syk-Rho GTPase signaling, and release of cytokines, via Syk-CARD9-Bcl10-MALT1 axis. B. CEACAM3-mediated phagocytosis and activation of neutrophils’ antimicrobial responses. N. gonorrhoeae binding to CEACAM3 receptor results in phosphorylation of its cytoplasmic ITAM motif by Src PTKs, and recruitment and activation of Syk kinase. Although Syk seems to be dispensable for engulfment of a single bacteria, it is essential for activation of oxidative burst and degranulation responses downstream of CEACAM3. CEACAM3 signaling also involves recruitment of Vav and PI3K, while Rac GTPases have been shown to be responsible for phagocytosis.

22 a contentious debate. At the onset of my studies, it was clear that the outcome of this interaction was dramatically different depending on whether gonococci expressed Opa proteins adhesins that engaged CEACAM3 or not, since this innate decoy receptor elicited an opsonin- independent engulfment and killing of the bacteria. Opa expression appears to be detrimental to gonococcal survival during encounter with PMNs, while bacteria that phase vary off their Opa proteins are able to both avoid phagocytosis and the neutrophils’ antimicrobial responses [195- 197,209,210,217]. However, considering the importance of Opa in epithelial attachment [77], and consistent isolation of Opa+ isolates from clinical samples [218-220], gonococcal ability to express Opa proteins seems essential for the bacteria to establish infection and for ongoing survival within tissue [210]. And while Opa-CEACAM3 interactions explain the close association of N. gonorrhoeae with neutrophil, it did not offer insight as to why there is such a profuse inflammatory response in the first place. I therefore sought to understand whether CEACAM3 might also contribute to the innate response and/or immunopathogenesis during N. gonorrhoeae infection. In Chapter 2, I take advantage of a transgenic mouse line expressing human CEACAM3, CEACAM5 and CEACAM6 to reveal that N. gonorrhoeae binding to CEACAM3 elicits a potent intracellular signaling cascade that leads to secretion of cytokines by neutrophils and consequently recruits other neutrophils to the infected tissues. As they encounter the gonococci within infected tissues, this next wave of neutrophils becomes similarly activated, leading to progressive expansion in phagocytic cell numbers until they overwhelm infected tissues. In Chapter 3, I dissect the signaling cascade that connects CEACAM3 to other pattern recognition receptors and the pro-inflammatory transcriptional program, and differentiate between phagocytic and inflammatory signaling pathways downstream of CEACAM3. In Chapter 4, I present work done in collaboration with Dr. Henry Wong, which revealed a marked in vivo selection for expression of phase variable Opa protein variants that bind CEACAM1 and/or CEACAM5 but not CEACAM3, consistent with CEACAM3-mediated neutrophil interactions being a detrimental outcome for the bacteria. This is the first study showing phenotypic selection for distinct CEACAM-binding phenotypes in vivo, and supports a model whereby binding to different CEACAMs can either facilitate infection or drive bacterial clearance within the genital tract. In Chapter 5, I bring my findings together into a model describing how CEACAM3 contributes to infection and immunopathogenesis, and explore the future implications of my work.

23 2 Global analysis of neutrophil responses to Neisseria gonorrhoeae reveals a self-propagating inflammatory program

This Chapter has been published as: Sintsova A, Sarantis H, Islam EA, Sun CX, Amin M, Chan CHF, Stanners CP, Glogauer M, Gray-Owen SD. Global analysis of neutrophil responses to Neisseria gonorrhoeae reveals a self-propagating inflammatory program. PLoS Pathog. 2014;10(9):e1004341.

Acknowledgements: Dr. Sarantis contributed to Figure 1-1A,C, F, G; EA Islam contributed to Figure 5-1E, F; CX Sun contributed to Figure 2-8A.

24 2.1 ABSTRACT

An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with Neisseria gonorrhoeae. Neutrophils undergo an aggressive opsonin-independent response to N. gonorrhoeae, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa+ bacteria. In this chapter, I sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an in vivo model of N. gonorrhoeae infection, I show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa- dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of N. gonorrhoeae infection.

25 2.2 INTRODUCTION

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a re-emerging global health concern, with over a hundred million cases diagnosed each year, the recent emergence of multi- drug resistant strains that have led to its ‘superbug’ status, and a lack of success in vaccine development [1,2,221]. Symptomatic infection with N. gonorrhoeae results in acute inflammation of the urogenital tract and a purulent urethral discharge consisting almost exclusively of neutrophils. If left untreated, gonococcal infection can lead to serious chronic conditions, such as pelvic inflammatory disease and infertility, which stem from an overzealous response to the infection [1]. N. gonorrhoeae is a Gram-negative diplococcus that is highly adapted to colonization of the human urogenital tract. The initial interaction between the bacteria and epithelia is mediated by type IV pili, which retract to allow tight association with the mucosal epithelia [17]. More intimate interactions are then facilitated by adhesins including the neisserial Opa proteins binding to certain epithelial cell-expressed members of the carcinoembryonic antigen-related adhesion molecule (CEACAM) family: CEACAM1, CEACAM5, and CEACAM6 [81,85,86 1997,87,88,222]. CEACAMs represent a subset of the Ig superfamily and consist of a variable number of Ig-like constant domains and an Ig variable domain-like N-terminus that allows Opa binding [223-225]. Attachment to apically-expressed CEACAMs is sufficient to trigger bacterial engulfment and transcytosis across the epithelia to allow entry into the subepithelial space [30,226]. CEACAM1 is notable among the family in that, in addition to being on epithelial cells, it is also expressed on certain endothelial, lymphocytic and myeloid cells. Bacteria exploit its co-inhibitory function, which depends upon its cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), to suppress T cell [45,89,90], B cell [227], dendritic cell [51] and epithelial cell [93] responses (reviewed in [37]). While binding to CEACAMs on most cell types tends to facilitate infection, Opa proteins may also bind to neutrophil-expressed CEACAM3. When this occurs, CEACAM3 triggers an efficient opsonin-independent phagocytosis of the bacteria [196,202,212]. Ligation of CEACAM3 also promotes a Syk kinase- and phosphatidylinositol-3-kinase-dependent recruitment and downstream activation of the neutrophils’ antimicrobial responses, including degranulation and oxidative burst [195,196,202,204,212-214,228]. These effects are driven by the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM), which distinguishes CEACAM3 from the other CEACAMs that N. gonorrhoeae binds. Considering that CEACAM3

26 is human-restricted, expressed on neutrophils and lacks cell adhesion function, CEACAM3 is now generally considered to be an innate immune receptor allowing capture and elimination of bacteria that colonize epithelial tissues via other CEACAMs [29,194,202,204,212]. Neutrophils are specialized for rapid transmigration to sites of infection in response to a variety of stimuli, including chemotactic gradients and presence of bacterial components. Following recruitment to the infected tissue, neutrophils effectively phagocytose opsonized bacteria, activate production of reactive oxygen species [229] and release toxic antimicrobial peptides and proteins from cytoplasmic granules [96,230]. Conventionally, neutrophils were thought to have little to no controlled expression of new gene products, depending mostly on constitutively-expressed proteins and pre-loaded granules assembled during maturation. In recent years, it has become evident that properly stimulated neutrophils respond by synthesizing new proteins [117,231,232], however surprisingly little is known about the control of gene expression. In this work, I show that heterologous expression of human CEACAMs in transgenic mouse neutrophils permits effective opsonin-independent neisserial binding and neutrophil activation in a manner reflecting that seen with human neutrophils. Moreover, I reveal that Opa- dependent CEACAM3 binding drives a potent neutrophil transcriptional response that elicits production of pro-inflammatory cytokines via a PKCδ and Tak1 serine/threonine kinase- dependent pathway triggered downstream of Syk tyrosine kinase. Furthermore, I observed that infection of human CEACAM-expressing transgenic mice with N. gonorrhoeae results in a dramatically higher neutrophil influx to the infection site when compared to wild-type mice. Together, this study establishes that bacterial binding to CEACAM3 effectively recruits more neutrophils to the infected tissues. While providing an effective strategy to combat the initial infection, this self-propagating cycle of events can also lead to the pathogenic inflammatory response that typifies symptomatic gonorrhea.

2.3 MATERIALS AND METHODS

2.3.1 Ethics Statement

All animal experiment procedures were approved by the Animal Ethics Review Committee of the University of Toronto (Approval #20010054 and #20010055), which is subject to the ethical and legal requirements under the province of Ontario’s Animals for Research Act and the federal Council on Animal Care.

2.3.2 Animals

Generation of the CEACAM1-humanized mouse line was previously described [233]. CEABAC2 mice, generated by stable integration of a human-derived bacterial artificial chromosome (BAC) encoding the human CEACAM3, CEACAM5, CEACAM6 and CEACAM7 genes, have been previously described [234]. Wild type (WT) mice used are littermates of the CEABAC2 animals.

2.3.3 Reagents and Antibodies

All reagents were from Sigma (Oakville, Ontario, Canada) unless otherwise indicated. The anti-gonococcal polyclonal rabbit antibody (UTR01) was described previously [227]. Rabbit anti- CEACAM polyclonal and normal serum was from Dako (Mississauga, ON). CEACAM pan-specific D14HD11, CEACAM1-specific 4/3/17 and CEACAM6-specific 9A6 antibodies were from Genovac (Freiburg, Germany), and the CEACAM3-specific Col-1 antibody was from Zymed (San Francisco, CA). Phospho-p38, p38, phospho-Erk1/2, Erk1/2, phospho-PKCδ and PKCδ-specific antibodies were form Cell Signaling Technology. The IκBα- specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent conjugates were from Jackson ImmunoResearch Laboratories (Mississauga, ON), except Texas red- phalloidin, which was from Molecular Probes (Eugene, OR). The Tak1 inhibitor ((5z)-7- oxozeaenol) was from Millipore (Billerica, MA), and the p38 inhibitor (SB203580) and the Src family kinase-specific PP2 were from Calbiochem (La Jolla, CA). Erk1/2 inhibitor (UT0126) was purchased from Cell Signaling.

2.3.4 Bacterial Strains

- + The isogenic Opa and Opa (Opa57, allows binding to CEACAM1, CEACAM3, CEACAM5, and CEACAM6) N. gonorrhoeae MS11 strains (N302 and N313, respectively;

28 [235]) were kindly provided by Dr. T.F. Meyer, and their phenotypes have been described previously [81].

2.3.5 Primary Neutrophil Isolation

Human neutrophils were isolated from citrated whole blood taken from healthy volunteers by venipuncture using Ficoll-Paque Plus (Amersham Biosciences; Buckinghamshire, England). Contaminating erythrocytes were removed by dextran sedimentation and hypotonic shock, as described previously [204]. Mouse bone marrow neutrophils were taken from 8 to 10-week old mice that were humanely euthanized by CO2 inhalation. Femurs and tibias were removed, and bone marrow was isolated and separated on a discontinuous Percoll gradient (80%/65%/55%) as described previously by others [236]. Neutrophils were recovered at the 80%/65% interface.

2.3.6 Human CEACAM Expression in CEABAC Neutrophils

WT and CEABAC neutrophils (106 cells) were lysed in boiling SDS buffer and CEACAMs detected using SDS-PAGE and immunoblots probed with indicated CEACAM- specific antibodies. For flow cytometry analysis of cell-surface CEACAM expression, 106 PMNs from CEABAC or WT littermates were spun down and fixed in 1% PFA in Hank’s Buffered Saline Solution (HBSS) prior to immunofluorescence staining.

2.3.7 Whole Cell Phosphorylation Assays

106 neutrophils per sample were infected with N. gonorrhoeae at multiplicity of infection of 10 in 250 µl of Hank’s buffered saline solution (HBSS) [237]. Infections were stopped by centrifugation at 2400 g for 3 min at 4oC, lysed in boiling SDS sample buffer, and boiled for a further 10 min. Samples were resolved by SDS-PAGE and immunoblotted.

2.3.8 Bacterial infections for immunofluorescence microscopy

5 5 x 10 WT or CEABAC bone marrow-derived PMNs were centrifuged onto washed mouse serum-coated coverslips at 1500 rpm for 10 min. Cells were infected at MOI of 25 (for binding and internalization studies) in a volume of 500 µl, re-centrifuged for 5 minutes at 500 rpm to facilitate bacterial association with cells, and then incubated at 37oC for indicated durations. Post-infection, samples were washed with Hank’s buffered saline solution HBSS, and fixed using 3.7% paraformaldehyde. Cells were stained for CEACAM, actin and bacteria and

29 observed as described previously [88]. Intracellular bacteria were differentiated from extracellular via exclusion of antibody prior to membrane solubilization, as described [204].

2.3.9 PMN Killing Assays

Killing assays were adapted from Ball et al. [209]. Briefly, adherent WT and CEABAC neutrophils were infected at an MOI=1. At indicated time points, cells were washed and incubated with protease inhibitors for 15 minutes prior to lysis with 1% saponin and plated on GC agar. Bacterial survival was evaluated relative to CFUs present at 0 time point.

2.3.10 Oxidative Burst and Degranulation Assays

For chemiluminescence-based oxidative burst assay, 5x105 cells were incubated with 25 µg/ml 5-amino-2,3-dihydro-1,4-phthalazinedione (‘luminol’; Sigma) in a volume of 100 µl, and then treated with agonists in a total volume of 200 µl, in triplicate. Infections proceeded for 60 min at 37oC, after which luminescence was read using a Tecan plate reader with i-control software. For flow cytometry-based degranulation assay (CD11b release), 106 PMNs were treated with agonists in 500 µl of HBSS for 30 minutes at 37oC. Infections were stopped by centrifugation at 2,400 g for 3 min at RT and cell pellets then fixed in 1% PFA before staining with 1.25 µg of PE-conjugated rat anti-mouse CD11b in a total volume of 50 µl. Myeloperoxidase (MPO), elastase, and lactoferrin release assays were performed essentially as described by others. Briefly, 106 PMNs were exposed to agonists in a total volume of 500 µl and then incubated for 30 minutes at 37oC. Cells were then pelleted and supernatants collected. For MPO assays, 50 µl of supernatant was mixed with 150 µl SureBlue tetramethylbenzidine peroxidase substrate (KPL; Gaithersberg, MD), and plates were read spectrophotometrically at 650 nm. For the elastase assay, 50 µl of supernatant was diluted 2-fold in PBS and incubated with 100 µl DQ elastin substrate conjugated to BODIPY FL (from the EnzCheck Elastase kit; Molecular Probes), and then incubated for 24 hours at RT before reading fluorescence with 488 nm excitation and 515 nm emission. For both MPO and elastase assays, a percentage (%) release is shown, calculated as the amount of the protein in the supernatant divided by the total amount in 106 CHAPS-lysed cells. Lactoferrin release from PMN granules was assayed by ELISA as described by others [238]. To induce release of primary granule components into medium, cells were pre-treated with 5 µM cytochalasin B for 5 minutes at 37oC prior to agonist treatment.

30 2.3.11 PMN Gene Array Experiments

CEABAC and WT bone marrow neutrophils (107 cells) were either infected with Opa+ N. gonorrhoeae (MOI=10) or left uninfected for 1 h. The infections were stopped by centrifugation at 2400 g for 5 min at 4oC. RNA was extracted and purified using the Qiagen RNeasy kit. Samples from 3 independent experiments were analyzed using an Illumina Mouse Whole Genome V2R2 array with 45,281 probes. The original data normalization and analysis were provided as a service by the Bioinformatics Department of the University Health Network [160] Microarray Centre, Toronto, ON. Data was analyzed using Genespring v11.0.1. 66 genes showed ≥ 2 fold change (FC) in gene expression in infected PMNs relative to uninfected controls. Gene lists were analyzed using the database for annotation, visualization, and integrated discovery (DAVID)[239], and manual examination. To compare WT vs. CEABAC neutrophil responses, we considered genes ≥1.5 FC in CEABAC over WT.

2.3.12 Cytokine Measurements

106 cells were infected with N. gonorrhoeae at MOI of 10 and incubated at 37oC for 3 h. Infections were then stopped by centrifugation at 2400 g for 5 min at 4oC, and supernatants were collected. Quantitative measurements of cytokines were performed using ELISA kits form R&D Systems (MIP-1α, KC and MIP-2) and BD Biosciences (TNF-α). For qRT-PCR, cells were infected as described above. At 3 h post infection, RNA was collected using Qiagen RNeasy kit and converted to cDNA using iScript RT Supermix (Bio-Rad Laboratories). qPCR was carried out using SsoAdvanced SYBR Green Supermix (Bio-Rad Laboratories). All transcript levels are shown as relative to those of GAPDH.

2.3.13 Chemotaxis Assay

Bone marrow neutrophils were isolated and suspended in HBSS with 1% gelatin (Sigma, G7041). A neutrophil suspension (1 x 106/mL) was allowed to attach to bovine serum albumin (BSA, Sigma A7906; 1 mg/ml)-coated glass coverslips (22 x 40 mm, Fisher 12-543-A) at 37°C for 10 minutes. The coverslip was inverted into a Zigmond chamber (Neuroprobe, z02) and 100 µL HBSS media was added to the right chamber with 100 µL HBSS media mixed with supernatants derived from infected CEABAC or WT neutrophils added to the left chamber. Time-lapse video microscopy was used to examine neutrophil movements in Zigmond chambers. Images were captured at 20-second intervals with a Nikon Eclipse E1000 Microscope

31 using the 40X objective. Cell-tracking software (Retrac version 2.1.01 Freeware) was used to characterize cellular chemotaxis from the captured images. Data comes from three independent experiments.

2.3.14 Air Pouch

CEABAC and WT littermate control mice (6-8 wk.) were anesthetized with isoflurane, and dorsal air pouches were raised by injecting 3 ml sterile air subcutaneously on days 0 and a further 2 ml on day 3. On day 5, the mice were anesthetized with isoflurane and injected with 1 ml PBS containing 2x106 cfu/ml of Opa- or Opa+ N. gonorrhoeae. Mice were sacrificed 6 h after the injection, and air pouches were then washed with 2 ml PBS. The cells present in the wash were counted with a hemocytometer and analyzed by Diff-Quick staining of the cytospins. The supernatants were analyzed by ELISA, as described above. In some cases, air pouch fibroblasts were obtained by instilling 0.05% trypsin containing 0.5 mM EDTA in DMEM (3 ml/pouch), as previously described [240]. These cells were seeded onto glass coverslips in a 24-well plate, cultured in DMEM-10% FBS with antibiotics overnight, and then infected with Opa+ N. gonorrhoeae the next day for 30 min. Cells were fixed and stained for immunofluorescence microscopy.

2.3.15 Neutrophil Depletion

Neutrophil depletion from mice was achieved by a single i.p. injection of 200 µl of sterile saline containing 250 µg of the Gr-1 specific monoclonal antibody RB6-8C5 at 24 h prior to infection. The RB6-8C5 hybridoma was generously provided by Professor Paul Allen, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis.

32 2.4 RESULTS

2.4.1 CEACAM-humanized transgenic mouse neutrophils respond to N. gonorrhoeae

Neisserial infection is exquisitely human-specific, with major receptors for the bacterial Opa protein adhesins being certain members of the human CEACAM family. While CEACAM homologues can be found in all vertebrates [241], only human CEACAMs have been observed to bind Neisseria. Mouse polymorphonuclear leukocytes (PMNs), which express mouse CEACAM1 on their surface, do not bind N. gonorrhoeae [212,242], whereas human PMNs specifically bind Opa-expressing but not Opa-deficient N. gonorrhoeae in an opsonin- independent fashion (Fig. 2-1A,C). Our lab has recently established that recombinant human CEACAMs encoded from constitutively expressed cDNA were functionally expressed in a mouse promyelocytic (MPRO) cell line [212]. Previous graduate student in our lab considered whether ectopic expression of intact human CEACAM genes in transgenic mouse neutrophils would also confer responsiveness to N. gonorrhoeae. To address this question, she performed experiments with bone marrow-derived neutrophils from human CEACAM-expressing CEABAC2 mice [234]. These mice were engineered using a BAC that encodes human CEACAM3, CEACAM5, CEACAM6 and CEACAM7, none of which have murine homologues. Using flow cytometric analysis and immunoblotting with CEACAM-specific antibodies, Dr. Sarantis confirmed that human CEACAM3 and CEACAM6 were expressed on the surface of CEABAC neutrophils, in a manner reflecting their expression on human neutrophils (Fig. 2-1A,B). When she exposed CEABAC neutrophils to N. gonorrhoeae expressing either the CEACAM-specific Opa+ or no Opa protein (Opa-), they effectively bound and engulfed the Opa+ but not Opa- bacteria, whereas no such association was apparent with wild type neutrophils regardless of Opa expression (Fig. 2-1C,D). I later showed that while the number of Opa+ bacteria captured by human CEACAM-expressing neutrophils was substantially (~10-fold) greater than what occurs with WT mouse neutrophils and/or Opa- bacteria (Fig. 2-1D), the bacteria that become engulfed are effectively killed regardless of whether or not human CEACAMs are involved in the uptake (Fig. 2-1E). These results differ from a recent study with human neutrophils which describe increased killing of Opa+ (relative to Opa-) bacteria [209], however it remains unclear whether this is a neutrophil species-dependent effect or result from differences in bacterial strain or methodology used in the two studies.

34 Figure 2-1. Human CEACAM expression by mouse neutrophils results in neisserial capture and internalization. (A) Human CEACAMs are expressed in CEABAC neutrophils in a manner reflecting that in human neutrophils. Human neutrophils [101], or wild type (WT) and CEABAC mouse neutrophils (bottom) were fixed and stained with antibodies specific for CEACAM1, CEACAM3, CEACAM6, or a mouse IgG isotype control, and analyzed by flow cytometry. Isotype histograms are shaded. (B) Immunoblot showing CEACAM expression in WT and CEABAC neutrophils. (C) Mouse neutrophils do not bind N. gonorrhoeae, while human neutrophils bind N. gonorrhoeae in an Opa-dependent manner. Mouse [101] or human (bottom) neutrophils were infected with non-opaque (Opa-) or Opa-expressing (Opa+) N. gonorrhoeae. Cells were visualized by staining filamentous actin with phalloidin [89], and bacteria are shown in green. Intracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy in (D). (E) WT and CEABAC PMNs kill Opa- and Opa+ bacteria with similar kinetics. Adherent WT and CEABAC PMNs were infected with either Opa- or Opa+ N. gonorrhoeae at an MOI=1. Bacterial survival over time was evaluated as CFUs present in PMN lysates at each time point relative to bacterial CFUs present at time 0. N=2. (F-G) N. gonorrhoeae infected CEABAC neutrophils respond analogously to human PMNs. Human (F) or mouse (WT and CEABAC) (G) neutrophils were infected with Opa- or Opa+ N. gonorrhoeae and oxidative burst and degranulation responses were analyzed as described in Materials and Methods.

35 2.4.2 N. gonorrhoeae infection activates CEABAC neutrophils

Previous work addressing individual CEACAM expression and its effect on neisserial infection was undertaken using transfected promyelocytic cell lines [212]. Because CEABAC transgenic neutrophils can bind and engulf N. gonorrhoeae (Fig. 2-1C), Dr. Sarantis wondered whether neutrophil-specific responses to N. gonorrhoeae were also reproduced in these cells. Human neutrophils respond to Opa+ N. gonorrhoeae by triggering an increased consumption of oxygen, resulting in the production of free oxygen radicals in the cell (the ‘oxidative burst’), as well as by releasing granule components to the cell surface or into the newly formed phagosome (‘degranulation’) [195]. Consistent with this, she observed that Opa+ N. gonorrhoeae were able to efficiently stimulate both the oxidative burst and release of primary and secondary granules (as determined by the release of neutrophil elastase and lactoferrin, respectively), in infected human neutrophils (Fig. 2-1F). In stark contrast, WT mouse neutrophils are surprisingly unresponsive to N. gonorrhoeae infection, illustrating the importance of human CEACAMs for these effects. However, in CEABAC neutrophils, Dr. Sarantis observed that the oxidative burst and degranulation were heightened in response to Opa+ bacteria (Fig. 2-1G), consistent with the function of Opa proteins in CEACAM binding.

2.4.3 N. gonorrhoeae drives an acute inflammatory program in neutrophils

While neutrophils were classically considered to be transcriptionally quiet, Fc receptor- mediated phagocytosis has long been known to promote IL-8 mRNA expression in neutrophils [229]. More recently, it has become clear that neutrophils have the capacity to become transcriptionally active in response to certain stimuli [117,231,232], yet neutrophil transcriptional responses to specific infections remain poorly understood. Consequently, to investigate whether N. gonorrhoeae might elicit a transcriptional response, I isolated RNA from uninfected and infected WT and CEABAC bone marrow-derived neutrophils and compared their transcriptional profile by full genome gene array 1 hour post-infection. DAVID functional annotation [239] and manual analysis of the results revealed that the general pattern of genes expressed in response to N. gonorrhoeae were similar in the WT and CEABAC animals, however, two categories of transcriptional up-regulation were apparent. In the first group are genes that are induced to a similar level in WT and CEABAC PMNs. Of these, the largest functional classes of genes are those involved in the regulation of inflammation (i.e. IL-10

36 Receptor α Subunit (il10ra); Suppressor of Cytokine Signaling 3 (SOCS3); Inhibitor of kappa B subunits (IκBδ, IκBζ)) and control of cell cycle and apoptosis (i.e. Bcl2, Gadd34, Gadd45) (Fig. 2-2A). In contrast, the CEABAC neutrophils displayed a marked up-regulation of acute inflammatory cytokine expression, including TNFα (2.1-fold induction over wild type neutrophils), IL-1α (2.7-fold induction) and the neutrophil chemoattractant and activators Gro- α/KC, MIP-1α and MIP-1β (1.5-, 3.3- and 3.5-fold induction) (Fig. 2-2B). It is pertinent to note that I detected no down-regulation of any gene expression at that time point. Considering that regulatory genes are expressed at similar levels between WT and CEABAC PMNs, while pro- inflammatory mediators are drastically higher in CEABAC cells, I infer that the cumulative effect of these changes in gene expression would be a heightened pro-inflammatory cytokine response when the gonococcal Opa proteins engage the neutrophil-expressed CEACAM3.

2.4.4 Opa-expressing N. gonorrhoeae drive production of pro- inflammatory cytokines in CEACAM-expressing neutrophils

To validate gene array results, and confirm that increases in transcript levels corresponded to PMN secretion of the protein products, I measured the production of MIP-1α, MIP-2, KC and TNFα protein in infected PMNs from WT and CEABAC mice (Fig. 2-3A). Significant (between 3- to-10 fold) increases in chemokine protein levels were observed in supernatants from CEABAC PMNs infected with Opa+ bacteria, compared to supernatants from CEABAC PMNs infected with Opa- bacteria. Critically, the WT neutrophil response was not affected by Opa expression, instead reflecting that seen with Opa- bacteria and CEABAC PMNs, demonstrating that both Opa and human CEACAMs are required for this effect. This increased chemokine secretion corresponded with increased levels of chemokine transcripts in these samples (Fig. 2- 3B), reflecting the data obtained via the gene array experiments, and establishing that de novo transcription is driving the cytokine response. Collectively, these data provide the first evidence of a neutrophil transcription-based inflammatory response to N. gonorrhoeae infection, and point to the CEACAM-Opa interaction as a critical driver of this inflammation.

Figure 2-2. N. gonorrhoeae drives an acute inflammatory transcriptional program. A gene array of WT and CEABAC PMNs infected with Opa+ N. gonorrhoeae was performed, and genes showing a significant increase in transcription over uninfected controls are illustrated. Genes with ≥ 2 fold induction over uninfected controls in both WT and CEABAC neutrophils 1 h post-infection are shown. (A) List of genes up-regulated to a similar level in both WT and CEABAC neutrophils relative to uninfected neutrophils. (B) List of genes differentially expressed between CEABAC and WT PMNs. The fold difference by which WT and CEABAC differ is indicated next to each bar.

38 2.4.5 CEACAM-Opa interaction drives an inflammatory response in human peripheral blood neutrophils

To confirm that the CEACAM- and Opa-dependent transcriptional response apparent in our transgenic mouse model reflected that occurring in human PMNs, peripheral blood neutrophils isolated from healthy volunteers were infected with Opa- or Opa+ N. gonorrhoeae and then subjected to quantitative RNA analysis. Opa+ N. gonorrhoeae-infected PMNs had substantially higher levels of MIP-1α, MIP-2, TNFα, and IL-1α transcript in all donors tested, when compared to Opa- infected controls (Fig. 2-3C). I therefore conclude that the CEACAM- Opa interaction potentiates the inflammation observed during human PMN infection, reflecting our findings with the CEACAM-humanized mouse model.

2.4.6 Phagocytosis and production of reactive oxygen species are not essential for inflammatory cytokine production

Unlike WT PMNs, CEABAC neutrophils efficiently bind and phagocytose Opa+ N. gonorrhoeae (Fig. 2-1C-D). This prompted me to consider whether uptake alone can account for the cytokine response of human CEACAM-expressing PMNs. To address this question, WT and CEABAC PMNs were pre-treated with cytochalasin D to inhibit phagocytosis prior to infection. While cytochalasin D pre-treatment led to a marked decrease in bacterial internalization (Fig. 2-4A); it had no effect on MIP-1α secretion (Fig. 2-5A). Furthermore, I used PMNs from a transgenic mouse line expressing human CEACAM1 but no CEACAM3. While the human CEACAM1-expressing neutrophils can efficiently phagocytose Opa- expressing N. gonorrhoeae [233], the neutrophils showed little chemokine response to infection (Fig. 2-4B). In considering that reactive oxygen species (ROS) have been linked to the activation of various inflammatory signals [243], I also sought to confirm whether ROS formed during the well-characterized oxidative burst response to Opa+ N. gonorrhoeae [195,210,212-214] explained the induced cytokine response. To this end, I prevented ROS production using the NADPH oxidase inhibitor diphenylene iodonium (DPI) and then measured cytokine production in WT and CEABAC neutrophils infected with Opa- and Opa+ bacteria. While DPI completely abolished the otherwise high levels of ROS produced upon exposure of CEABAC PMNs to Opa+ N. gonorrhoeae (Fig. 2-4C), this had no affect the chemokine response of CEABAC PMNs (Fig. 2-5A).

Figure 2-3. Opa-expressing N. gonorrhoeae drive CEACAM-dependent production of pro-inflammatory cytokines in neutrophils. (A-B) CEABAC PMNs secret pro- inflammatory cytokines in response Opa+ N. gonorrhoeae. Mouse (WT and CEABAC) neutrophils were infected with either Opa- or Opa+ N. gonorrhoeae (MOI 10). MIP-1α, MIP- 2, KC and TNFα production was measured 3 h post infection at (A) protein and (B) mRNA level. N≥3, error bars represent SEM. (C) Human neutrophils infected with Opa+ N. gonorrhoeae show increased levels of MIP-1α, MIP-2, TNFα, and IL-1α mRNA relative to PMNs infected with Opa- bacteria. Each symbol represents an individual donor. Cytokine mRNA levels shown as relative to levels of GAPDH mRNA. One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. For (A) and (B) stars indicate significance against all other conditions.

41 Figure 2-4. Phagocytosis and production of reactive oxygen species are not essential for inflammatory cytokine production. (A) Cytochalasin D treatment prevents bacterial uptake by CEABAC PMNs. WT and CEABAC PMNs were left untreated or pre-incubated with cytochalasin D (10 mg/ml) for 10 min prior to infection with either Opa- or Opa+ N. gonorrhoeae (MOI 10). Extracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy. (B) Human CEACAM1-expressing PMNs do not induce inflammatory cytokine production in response to Opa+ N. gonorrhoeae. Mouse (WT, CEABAC, and TG418) neutrophils were infected with either Opa- or Opa+ N. gonorrhoeae (MOI 10). MIP-1α and MIP-2 was measured 3 h post infection. Data shown as fold induction by Opa+ infection over Opa- infection. N=3. (C) DPI treatment abolishes oxidative burst response in CEABAC PMNs infected with Opa+ N. gonorrhoeae. WT and CEABAC PMNs were left untreated or pre-incubated with DPI (10 µM) for 30 min prior to infection with either Opa- or Opa+ N. gonorrhoeae (MOI 10). Oxidative burst was measured with as described in Materials and Methods. (D) Inhibition of Erk1/2 kinases does not affect inflammatory cytokine production. Erk1/2 inhibition does not affect cytokine production by CEABAC PMNs. WT and CEABAC PMNs were left untreated or pre-incubated with Erk1/2 inhibitor (UT0126, 10 µM) for 30 min prior to infection with Opa+ N. gonorrhoeae (MOI 10). MIP-1α production was measured 3 h post infection, N=3.

42 Taken together our data suggest that Opa protein-dependent engagement of CEACAM3 drives a cytokine response that is independent of its ability to promote bacterial phagocytosis and is not mediated by the ROS produced in response to infection.

2.4.7 CEACAM-Opa interaction drives activation of NF-κB and MAPK signaling

A large proportion of genes identified in the gene array study (MIP-1α, MIP-2, TNFα, IL-1α) are known to be activated by NF-κB. NF-κB transcription factors are major mediators of inflammation and have been shown to stimulate transcription of pro-inflammatory cytokines in response to LPS in PMNs [237] [244] [245]. NF-κB also governs transcriptional responses downstream of various ITAM receptors in other (non-neutrophil) cell types [246], including the innate immune receptor Dectin-1[247]. This led me to investigate NF-κB activation downstream of CEACAM3. Consistent with NF-κB being a downstream effector of CEACAM3, IκBα was degraded more rapidly and completely in CEACAM-expressing PMNs than in the WT cells (Fig. 2-5B). The p38 mitogen associated kinase (MAPK) has been shown to act along side NF-κB in the activation of PMN transcriptional responses to LPS [248] [244]. Consequently, I considered whether p38 might also be involved in the CEACAM-mediated transcriptional response. Infected CEABAC neutrophils exhibit increased levels of p38 phosphorylation when compared to WT (Fig. 2-5C), indicating that they are more effectively activated in response to N. gonorrhoeae infection. In contrast, the Erk1/2 MAPKs were not phosphorylated in a CEACAM- dependent manner (Fig. 2-5D), suggesting selective activation of p38 kinase upon CEACAM ligation. To assess whether p38 activity contributed to the CEACAM-dependent cytokine response, PMNs were exposed to a p38-specific inhibitor (SB203580) prior to infection. This treatment effectively blocked production of MIP-1α and MIP-2 in CEABAC PMNs infected with Opa+ N. gonorrhoeae (Fig. 2-5E), while inhibition of Erk1/2 phosphorylation had no effect on cytokine secretion (Fig. 2-4D). Together, this data supports an essential role for p38 MAPK in the CEACAM-dependent transcriptional response. It has been reported that the mitogen-activated kinase kinase kinase (MAPKKK) family member TAK1 can activate both p38 MAPKs and NF-κB [249]. To test the involvement of TAK1 in the p38 activation observed during neisserial infection of PMNs, I used the TAK1 inhibitor (5z)-7-oxozeanol. TAK1 inhibition effectively abrogated MIP-1α and MIP-2 secretion

44 Figure 2-5. N. gonorrhoeae infection elicits an NF-κB and p38 MAPK signaling- dependent cytokine response in CEABAC neutrophils. CEABAC-Opa interaction leads to activation of NF-κB signaling. (A) WT and CEABAC PMNs were left untreated or pre- incubated with DPI (10 µM) or cytochalasin D (10 mg/ml). PMNs were then infected with either Opa- or Opa+ N. gonorrhoeae and MIP-1α levels were measured 3 h post infection. N=3, error bars represent SEM. One-Way ANOVA indicates no significant differences between corresponding samples. (B) WT and CEABAC PMNs were infected with Opa+ N. gonorrhoeae (MOI=10 bacteria/PMN) for times indicated, and levels of IkBα were then detected by immunoblot with IκBα antibody. Assessment of tubulin levels confirmed equal protein loading. (C, D) CEABAC-Opa interaction promotes activation of p38 MAPK. WT and CEABAC PMNS were infected as in (B). Activation of MAP kinases p38, Erk1, and Erk2 was determined by immunoblot with phospho-specific antibodies. Total p38 and Erk1/2 levels were assessed to ensure equal protein loading. (D, F) WT and CEABAC PMNs were left untreated or preincubated with (D) p38 inhibitor (SB203580, 10 µM) or (E) TAK1 inhibitor ((5z)-7-oxozeaenol, 500 nM). PMNs were then infected with either Opa- or Opa+ N. gonorrhoeae and MIP-1α and MIP-2 levels were measured 3 h post infection. N≥3, error bars represent SEM. One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. Stars indicate significance against all other conditions.

45 by the Opa+ N. gonorrhoeae-infected CEABAC neutrophils (Fig. 2-5F). Importantly, neither bacterial adherence nor phagocytosis by the PMNs were affected by either the p38 or TAK1 inhibitors (Fig. 2-6E, F), confirming that the effect of these two compounds on cytokine expression was not due to their inhibition of these cellular processes.

2.4.8 SFK, Syk, and PKCδ couple CEACAM3 to the downstream transcriptional response

CEABAC neutrophils express both human CEACAM3 and CEACAM6. Both receptors facilitate bacterial uptake, yet previous work has shown that the activation of neutrophil bactericidal processes, including degranulation and oxidative burst, occur via CEACAM3 alone [195,212]. We sought to confirm whether the pro-inflammatory response observed in CEABAC neutrophils was solely mediated by CEACAM3, and to determine whether the inflammation depended upon the CEACAM3 ITAM-dependent signaling. Since CEACAM3, unlike the GPI- anchored CEACAM6, relies on phosphorylation of its cytoplasmic ITAM by Src family kinases (SFK) for its activation, I exploited the SFK-specific inhibitor PP2 that has previously been shown to block CEACAM3 ITAM-dependent signaling [195,196,203,207]. Inhibition of SFK significantly abrogated cytokine production (Fig. 2-6A), but did not affect bacterial adherence or phagocytosis (Figs. 2-6E, F). Considered together, the data suggests that CEACAM3 ITAM phosphorylation is essential for induction of pro-inflammatory response. This also indicates a divergence in the bacterial engulfment and transcriptional pathways, since the tyrosine phosphorylation-independent CEACAM3- and CEACAM6-mediated engulfment of N. gonorrhoeae [212] is not, itself, sufficient to elicit the pro-inflammatory response. Work in other systems has shown that Syk kinase is an essential mediator of ITAM-mediated responses in general [250] and CEACAM3-specific neutrophil responses in particular [212], though there is some suggestion that CEACAM3 can signal independent of Syk [197]. Therefore, I tested whether Syk contributed to CEACAM3-dependent inflammatory signaling using the specific inhibitor, piceatannol. Inhibiting Syk function led to a significant reduction in chemokine production by CEABAC neutrophils (Fig. 2-6B), implicating a critical role for this kinase in the CEACAM3-dependent pro-inflammatory cytokine responses. Recently, the serine/threonine kinase PKCδ was shown to link signaling from the ITAM- containing innate immune receptor Dectin-1 to NF-κB activation in dendritic cells [180]. Consequently, I assessed PKCδ activation during N. gonorrhoeae infection of WT and/or CEABAC PMNs. CEABAC neutrophils showed substantially increased phosphorylation of

47 Figure 2-6. CEACAM3 signaling is required for the PMN cytokine response to N. gonorrhoeae. Inhibition of Src-family kinases and Syk leads to decreased cytokine production by infected PMNs. (A, B) WT and CEABAC PMNs were left untreated or pre- incubated with (A) Src-family kinase inhibitor (PP2, 10 µM), or (B) Syk inhibitor (piceatannol, 50 µM). PMNs were then infected with either non-opaque (Opa-) or Opa- expressing (Opa+) N. gonorrhoeae, and MIP-1α and MIP-2 levels were measured 3 h post infection. N≥3, error bars represent SEM. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (C) Opa+ N. gonorrhoeae infection leads to phosphorylation of PKCδ. WT and CEABAC PMNs were infected with Opa+ N. gonorrhoeae (MOI=10 bacteria/PMN) for times indicated times. PKCδ activation was measured by immunoblot using phospho-PKCδ antibody. Immunoblot for PKCδ indicates equal protein loading. (D) CEABAC PMNs were left untreated or pre-incubated with PKC inhibitor (BIS II, 10 µM). PMNs were then infected with Opa- or Opa+ N. gonorrhoeae, and MIP-1α levels were measured 3 h post infection. One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. Unless otherwise indicated, stars indicate significance against all other conditions. (E, F) Inhibition of SFK, Syk, TAK1 or p38 does not affect bacterial binding (E) or phagocytosis (F). WT and CEABAC PMNs were left untreated or pre-incubated with indicated inhibitors. PMNs were then infected with Opa+ N. gonorrhoeae (MOI=25) for 30 min. Intracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy. (G) Schematic representation of proposed signaling pathway resulting in bacterial engulfment, activation of oxidative burst and degranulation, and cytokine production.

48 PKCδ relative to that seen in WT PMNs (Fig. 2-6C), suggesting it is also activated downstream of CEACAM3. Furthermore, pretreatment of CEABAC PMNs with the PKC inhibitor bisindolylmaleimide II (BIS II) led to the inhibition of MIP-1α production in response to Opa+ N. gonorrhoeae (Fig. 2-6D), consistent with PKCδ actively contributing to the CEACAM3- driven inflammatory response. In summary, I have outlined a novel signaling cascade downstream of CEACAM3 that is distinct from CEACAM3-mediated bacterial engulfment and activation of antimicrobial responses (Fig. 2-6G), and potentially contributes to the excessive inflammatory response typical of N. gonorrhoeae infection.

2.4.9 CEACAM-Opa intensifies the inflammatory response in vivo

The heightened expression of pro-inflammatory cytokines by CEACAM-humanized PMNs should result in increased neutrophil chemotaxis to the site of N. gonorrhoeae infection, an outcome consistent with the clinical manifestations of gonorrhea. To test this, I collected supernatants from WT and CEABAC PMNs infected with Opa+ N. gonorrhoeae, and our collaborator in Dr. Glogauer’s laboratory, Dr. Sun, measured their ability to effect the speed and directionality of neutrophils using a Zigmond chamber. Consistent with the increased expression of chemotactic factors when CEABAC neutrophils are infected with N. gonorrhoeae, the chemotaxis of uninfected neutrophils was significantly greater in response to culture supernatants from the N. gonorrhoeae-infected CEABAC neutrophils, as compared to supernatants from infected WT neutrophils (Fig. 2-7A). To understand the implications of these effects during in vivo infection, I measured the relative contribution of neutrophil CEACAM3 on inflammation using a subcutaneous air-pouch model, which allows the effective recovery and analysis of leukocyte recruitment to an otherwise sterile site [251]. Opa expression did not affect leukocyte recruitment into air pouches formed in WT mice. However, in CEABAC mice, the Opa+ gonococci elicited a significantly increased infiltration of neutrophils relative to that seen in response to Opa- bacteria (Fig. 2-7B). Giemsa- Wright staining of the CEABAC-Opa+ air pouch infiltrate showed that nearly all of the cells (>95%) present are PMNs (Fig. 2-7C), with many neutrophils containing intracellular gonococci. Notably, the number of PMNs present in CEABAC mice infected with Opa- bacteria reflected that seen in the WT mice, indicating that both Opa and human CEACAMs are required to drive the more intense inflammatory response.

50 Figure 2-7. CEACAM binding stimulates the inflammatory response to N. gonorrhoeae in vivo. (A) Neutrophil migration assay. PMN migration speed towards N. gonorrhoeae infected CEABAC or WT neutrophil-derived supernatants was measured using a Zigmond chamber. One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, ***P<0.001 (B) Neutrophil infiltration is more pronounced in human CEACAM-expressing mice in an infected subcutaneous air pouch model. Manual neutrophil counts of wash fluids collected from air pouches. ‘PBS’ denotes mice injected with sterile PBS. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (C) Giemsa-Wright stain of wash fluid collected from CEABAC air pouch infected with Opa+ N. gonorrhoeae. The arrows point towards N. gonorrhoeae inside the neutrophil. (D) Neutrophil-expressed CEACAMs mediate N. gonorrhoeae binding within the air pouch. Cells from trypsinized air pouches were grown on coverslips in the presence of antibiotics, and infected in vitro with Opa+ N. gonorrhoeae. Cells were visualized by staining for filamentous actin [89], CEACAMs (green), DNA (blue) and bacteria (cyan). (E) Levels of MIP-1α, MIP-2, KC, and IL-1β were measured in wash fluids collected from air pouches. PMN- refers to mice in which neutrophils were depleted by administration of the Gr1-specific clone RB6-8C5 antibody one day prior to infection with Opa+ N. gonorrhoeae. One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (F) Inhibition of pro-inflammatory signaling reduces neutrophil infiltration into the air pouch. Neutrophil counts of wash fluids collected from CEABAC mice infected with Opa+ N. gonorrhoeae in the presence or absence of TAK1 inhibitor.

51 Considering the marked increase in neutrophil response in the CEABAC animals, we considered whether it was possible that cells lining the air pouch could differentially associate with N. gonorrhoeae, which would contribute to the inflammatory milieu. To test this, I administered trypsin/EDTA into an uninfected air pouch from CEABAC mice, harvested the cells that were released, and then seeded them onto glass coverslips. The next day, cells were infected with Opa+ bacteria, and bacterial binding and CEACAM expression were assessed using immunofluorescence microscopy. Cells lining the pouch did not express CEACAM and did not bind or take up the N. gonorrhoeae (Fig. 2-7D, top). When the same experiment was conducted on air pouches infected with Opa+ N. gonorrhoeae, I observed a large number of PMNs had infiltrated the air pouch lining. As expected, these PMNs expressed high levels of the human CEACAMs, and effectively bound the bacteria, unlike the adjacent fibroblast lining (Fig. 2-7D, bottom). These results are consistent with the enhanced inflammation being due to differential association of the gonococci with the CEACAM3-expressing neutrophils, without obvious effect on their interaction with the surrounding tissues. The air pouch experiments suggest that the CEACAM-dependent association between N. gonorrhoeae and the resident neutrophils promotes subsequent neutrophil recruitment, presumably due to the establishment of a chemotactic gradient. Consistent with this, the pro- inflammatory chemokines MIP-1α, MIP-2, KC, and IL-1β were all increased in the washes from Opa+-N. gonorrhoeae infected air pouches in CEABAC mice relative to that seen in the infected WT littermates (Fig. 2-7E). To address whether neutrophils directly contribute to the higher levels of cytokines observed, I infected mice that had been depleted of neutrophils by administration of the Gr1-specific RB6-8C5 antibody prior to the introduction of N. gonorrhoeae. The levels of MIP-1α and IL-1β were significantly lower in the air pouches from PMN-depleted mice, indicating that neutrophils are the primary source for both of these chemokines (Fig. 2-7E). Interestingly, the levels of KC and MIP-2 were dramatically higher in PMN-depleted mice, indicating that these chemokines are produced by cells other than PMNs under these conditions. While these chemokines are both produced by CEABAC neutrophils in response to N. gonorrhoeae (Fig. 2-3), they can also be produced by a variety of tissues [96], including synovial fibroblasts [252]. Considering that their levels increased upon neutrophil depletion, I interpret the increased response to suggest a delay in bacterial clearance in the absence of PMNs. The increased IL-1β and MIP-1α in CEABAC mice thereby reflect a local neutrophil response whereas MIP-2 and KC levels appear to be the cumulative effect of both neutrophil and underlying tissue responses.

52 To link the CEACAM3-dependent intracellular response evident from my cell-based experiments with inflammation in vivo, I repeated the air pouch experiment, this time assessing the effect of the TAK1 inhibitor, which was administered to mice 1 h prior to infecting the air pouch. Consistent with my model that the inhibition of CEACAM3 signaling would suppress the inflammatory response to N. gonorrhoeae, I observed a decrease in the number of infiltrating PMNs upon administration of the TAK1-inhibitor relative to the untreated animals (Fig. 2-7F).

53 2.5 DISCUSSION

The picture of neutrophils as ever-ready weapons of defense aiming to achieve efficient pathogen clearance has become an axiom. While still true, recent evidence suggests that they have the ability to nuance their response through de novo gene expression in response to certain microbial cues. In addition to their classical role in direct microbial killing, neutrophils can produce a range of cytokines with the potential to affect inflammation through the activation and induced chemotaxis of various leukocytes [117,253]. However, their specific contribution to the cytokine milieu and inflammatory response remains underappreciated in vivo. The emerging picture of neutrophils as a dynamic, responsive cell population has important implications for our understanding of the overzealous neutrophil response that typifies gonorrhea. Our findings reveal that the decoy receptor CEACAM3, in addition to facilitating the effective capture and killing of N. gonorrhoeae, also helps drive inflammation. While recruitment of more PMNs to combat infection would seem to be an effective innate immune strategy during early infection, the persistent exposure of CEACAM3-expressing PMNs to Opa+ gonococci can promote a self- perpetuating and, ultimately, pathogenic response such as is associated with gonorrhea or pelvic inflammatory disease. In this work, I demonstrate that human CEACAM-expressing transgenic mouse PMNs respond to N. gonorrhoeae in a manner that parallels those of human PMNs. Unlike neutrophils from WT mice, the CEABAC neutrophils undergo a vigorous oxidative burst and degranulation response to N. gonorrhoeae. Since the pathology associated with gonococcal disease primarily arises due to tissue damage caused by the recruited neutrophils, I considered whether CEACAM-dependent interactions with the bacteria could also contribute to the inflammatory response. I observed that N. gonorrhoeae binding to human CEACAM3 leads to the acute activation of a pro-inflammatory transcriptional program that results in the production of the inflammatory mediators such as MIP-1α, MIP-2, KC and TNFα. While relatively little is known about signal transduction in PMNs relative to other cell types, Opa binding to CEACAM3 elicits signaling via a pathway closely reminiscent of that triggered downstream of the innate antifungal receptor Dectin-1 [150,194,254]. As with Dectin-1, SFK and Syk kinase represent the first effectors downstream of CEACAM3, and their activity is required for the PMN oxidative burst and degranulation responses to Opa+ gonococci [212]. While Syk was shown to serve a regulatory role in the context of PMNs [255], I have observed that Syk contributes to the inflammatory cytokine response to N. gonorrhoeae by eliciting the PKCδ and TAK1-dependent

54 activation of NF-κB. This CEACAM3-dependent expression of MIP-1α, MIP-2 and KC stimulates chemotaxis of uninfected neutrophils so as to augment their recruitment to the infected tissues, an effect that has the potential to contribute to both innate defense and the massive neutrophil recruitment that typifies gonorrhea (Fig. 2-8). The trade-off of having a direct link between CEACAM3 and inflammation is that, when uncontrolled, this response results in the ongoing infiltration of neutrophils, leading to permanent tissue damage such as is observed with N. gonorrhoeae-associated fallopian tube scarring and pelvic inflammatory disease. Supporting this model, I observed CEACAM- dependent increases in neutrophil recruitment into the gonococcal-infected tissues, brought on by the CEACAM3-dependent production of chemokines with the potential to promote continuous infiltration of neutrophils. Thus, CEACAM3 activation serves as a double-edged sword, promoting the immunopathology of gonorrhea through an over-activated immune response that is meant to clear the bacteria causing the infection. While the complete ablation of neutrophil recruitment would increase the bacterial burden, a better outcome would be to limit the chemokine response without blocking bacterial binding and phagocytosis (Fig. 2-8). As a proof of concept for this, I used a TAK1 inhibitor in vivo to block the pro-inflammatory response and, thereby, the influx of neutrophils without affecting their phagocytic capacity. Satisfyingly, this treatment decreased PMN recruitment to that seen in WT mice, effectively eliminating the CEACAM3-dependent inflammation in response to N. gonorrhoeae. It has been reported previously that the majority of neisserial isolates from infected patients have the capacity to bind to CEACAMs (Virji, 1996, 929–939). This may seem contradictory when considering the negative outcome for bacteria that bind to CEACAM3 on neutrophils. However, it is important to consider that, in contrast to CEACAM3, neisserial binding to other human CEACAMs facilitates attachment to mucosal epithelia [30,200,201,256] and suppression of both innate [93] and adaptive [45,89,90] immune responses, activities that are central to the establishment and persistence of infection. In this respect, it is curious to contrast CEACAM1, which is the evolutionary precursor of the CEA family, with the evolutionarily 'new' CEACAM3. While CEACAM1 is present in all vertebrates and is broadly expressed on many cell types, CEACAM3 can only be found in humans and is only expressed by neutrophils. When this is considered along with the fact that CEACAM3 possesses no cell adhesion function yet has a sufficiently conserved extracellular domain that can be engaged by the neisserial Opa proteins, we and others have suggested that

55 CEACAM3 functions as a decoy receptor that allows the capture and killing of CEACAM- targeting microbes. The apparent involvement of CEACAM3 in pro-inflammatory signaling establishes a role for neutrophils beyond basic microbial killing, and places CEACAM3 as both a potential contributor to the accelerated response to N. gonorrhoeae infection and, subsequently, to the immunopathology associated with the gonococcal disease. The evolutionary advent of CEACAM3 thus reflects the latest step in the ongoing dance between Neisseria and their only natural host, providing a snare that mobilizes our most potently bactericidal cells against this stealthy invader.

Figure 2-8. CEACAM3-mediated inflammation promotes immunopathology associated with N. gonorrhoeae infection. Upon in vitro infection, CEACAM3 allows efficient bacterial phagocytosis and clearance via activation of neutrophil antimicrobial responses including degranulation and oxidative burst. Concomitantly, CEACAM3 promotes de novo transcription of pro-inflammatory cytokines by the neutrophil (left panel). In vivo, this cytokine response recruits more neutrophils to the site of infection. As they arrive, these neutrophils will become activated via N. gonorrhoeae binding to CEACAM3, driving a self-propagating inflammatory process leading to the immunopathology associated with gonococcal disease (top right panel). By limiting the CEACAM3-dependent inflammatory cascade without affecting bacterial engulfment, such as via Tak1 inhibitor used in this study, bacterial clearance can continue while inflammation is reduced (bottom right panel).

57 3 Bcl10 regulates the synergistic inflammatory response elicited by CEACAM3 and TLR4

This Chapter is being prepared as a manuscript as: Sintsova A, Sarantis H, Mak, T, Glogauer M, Gray-Owen SD. Bcl10 regulates the synergistic inflammatory response elicited by CEACAM3 and TLR4.

Acknowledgements: Dr. Sarantis contributed to Figure 3-2A,B

3.1 ABSTRACT

The human-restricted innate immune receptor CEACAM3 functions as a decoy to capture microbes intent on using other CEACAM family members for adhesion to the mucosa. Bacterial binding to CEACAM3 leads to the Src family kinase-dependent phosphorylation of its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM), which promotes bacterial engulfment and an NF-κB-dependent cytokine transcriptional response by human neutrophils. Herein, I report that CEACAM cross-linking is not sufficient for induction of cytokine production, and show that the inflammatory response induced by N. gonorrhoeae infection is defined by integration of signals from CEACAM3 with endotoxin-specific TLR4. Using PMNs from a human CEACAM-expressing mouse line (CEABAC), I reveal a molecular bifurcation of the CEACAM3-mediated antimicrobial and inflammatory responses. Ex vivo experiments with CEABAC Rac2-/-, Bcl10-/- and MALT1-/- PMNs indicate that these effectors are not necessary for gonococcal engulfment, and only Rac2 contributes to the CEACAM3-mediated oxidative burst, yet all three effectors are required for CEACAM3-mediated cytokine production. Bcl10 and MALT1 are often considered to function as a complex, however, while Bcl10 contributes to the synergy between TLR4 and CEACAM3, MALT1 does not. Together, these findings reveal an integration of the specific innate immune receptor CEACAM3 with more conventional pattern recognition receptor signaling, providing a mechanism by which the innate immune system can escalate its response to a relentless pathogen.

59 3.2 INTRODUCTION

Symptomatic infection with N. gonorrhoeae, which causes the sexually transmitted disease gonorrhea, is characterized by a urethral or cervical exudate resulting from a massive influx of neutrophils to the infected mucosa. The recruited neutrophils effectively engulf and destroy gonococci in an opsonin-independent manner [87,196]. Activation of these phagocytic and antimicrobial pathways is dependent on neutrophil engagement by the phase-variable colony opacity-associated (Opa) adhesins expressed by N. gonorrhoeae, while a large proportion of gonococci lacking Opa-expression are able to survive neutrophil encounter [87,195,196,209,210,212-214]. Although some Opa variants bind to heparan sulfate proteoglycans, the majority targets specific members of the human CEACAM receptor family. CEACAMs are a large family of proteins that function in cell-cell adhesion, with downstream effects on cellular activation, proliferation, differentiation and cell survival [33,37]. N. gonorrhoeae has co-opted these receptors to facilitate infection, allowing Opa protein-mediated attachment to epithelial-expressed CEACAM1, CEACAM5 and CEACAM6 [37,81,87,88]. Rather than providing a simple anchor, CEACAM binding facilitates gonococcal entry into the sub-epithelial space by promoting transcytosis through the polarized epithelial layer [30]. Moreover, the Opa-dependent engagement of CEACAM1 suppresses the activation of both T cells [45,89,90] and dendritic cells [37,51] so as to help suppress the adaptive immune response. While binding to other CEACAMs facilitates infection [201,257], CEACAM3 is a decoy receptor that allows human neutrophils to phagocytose and eliminate pathogens with CEACAM-specific adhesins [195,197,205,212]. Expression of CEACAM3 in non-phagocytic cell lines is sufficient to allow opsonin-independent bacterial uptake via a process analogous to conventional Fc receptor-mediated phagocytosis [203,204]. As with FcγRIIA-mediated phagocytosis [151], CEACAM3-mediated bacterial uptake is dependent on phosphorylation of the receptor’s cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM), and is characterized by formation of actin-rich phagocytic cups triggered by GTP loading of Rac GTPase [88,197,203,205,207]. CEACAM3-driven engulfment is followed by reactive oxygen species (ROS) production, which is dependent on Syk kinase, PI3K, and the rapid translocation of NADPH oxidase components to gonococci-containing phagosomes [195,213,214]. CEACAM3 engagement also leads to release of both primary and secondary granule contents into the phagolysosomes, and treatment with Src or Syk kinase inhibitors prevents this degranulation response and improves bacterial survival [195,210,212,213].

60 In addition to promoting bacterial engulfment and destruction, I recently observed that CEACAM3 engagement by N. gonorrhoeae leads to production of potent pro-inflammatory cytokines, which drives further PMN recruitment in a self-perpetuating inflammatory cascade [258]. As with the anti-microbial response, this inflammatory response requires phosphorylation of the CEACAM3 ITAM tyrosines and subsequent recruitment of Syk kinase [258], which elicits PKCδ- and TAK1-dependent activation of an NF-κB-driven transcriptional response. By eliciting the expression of MIP-1α, MIP-2 and KC (mouse homologues of human IL-8), this stimulates the chemotactic recruitment of uninfected neutrophils to the infected tissues, an effect that has the potential to contribute to innate defense while also precipitating the massive neutrophil recruitment that typifies gonorrhea [258]. Herein, I report that the outcome of CEACAM3 binding depends upon context, since simple ligation of the receptor does not replicate the transcriptional response evident upon bacterial binding. Instead, CEACAM3 signals synergize with those from the endotoxin-specific pattern recognition receptor (PRR), TLR4, to elicit an inflammatory response more robust than that caused by either receptor alone. Since PRR engagement is not required for microbial engulfment by CEACAM3, the convergence of CEACAM3 and TLR4 signals must occur after the events that trigger phagocytosis. By breeding the human CEACAM-expressing CEABAC transgene into mouse lines with deficiency in downstream immune effectors, I reveal Rac2- deficient neutrophils engulf but do not undergo an oxidative burst or inflammatory response to N. gonorrhoeae, whereas Bcl10-deficiency and MALT-1 deficiency allow ROS production without a severely compromised cytokine response. Bcl10 deficiency, but not that of MALT-1, also led to disruption of collaborative cytokine production by TLR4 and CEACAM3, and the fact that Bcl10 did not contribute to either CEACAM3-mediated phagocytosis or the neutrophil response to purified LPS suggests that it is a key effector driving the inflammatory synergy between these two potent signaling pathways.

61 3.3 MATERIALS AND METHODS

3.3.1 Ethics Statement

3.3.2 Animals

CEABAC10 and CEABAC2 (10 and 2 transgene copies, respectively) FvB mice, generated by stable integration of a human genome-derived bacterial artificial chromosome (BAC) encoding the human CEACAM3, CEACAM5, CEACAM6 and CEACAM7 genes, have been previously described [234]. Wild type (WT) mice used are littermates of the CEABAC animals. These CEABAC mice were backcrossed for 12 generations onto the C57Bl/6 background, and then interbred with Rac2-/- mice [236], Bcl10-/-, and MALT1-/- [259,260] mice to generate the CEABAC Rac2-/-, CEABAC Bcl10-/-, and CEABAC MALT1-/- lines.

3.3.3 Reagents and Antibodies

3.3.4 Bacterial Strains

The isogenic transparent (Opa-) and Opa+ (expressing the CEACAM1, CEACAM3,

CEACAM5 and CEACAM6 binding Opa57) N. gonorrhoeae MS11 strains (N302 and N313, respectively; [235]) were kindly provided by Dr. T.F. Meyer, and their phenotypes have been described previously [81].

3.3.5 Primary Neutrophil Isolation

62 shock, as described previously [204].

Mouse bone marrow-derived neutrophils were taken from 8- to 10-week old mice that were humanely euthanized by CO2 inhalation. Femurs and tibias were removed, and bone marrow was isolated and separated on a discontinuous Percoll gradient (80%/65%/55%) as described previously by others [236]. Neutrophils were recovered at the 80%/65% interface.

3.3.6 Bacterial infections for immunofluorescence microscopy

5 x 105 WT or CEABAC mouse bone marrow-derived PMNs were centrifuged onto washed mouse serum-coated coverslips at 1500 rpm for 10 min. Cells were infected at an MOI of 25 (for binding and internalization studies) in a volume of 500 µl, re-centrifuged for 5 minutes at 500 rpm to facilitate bacterial association with cells, and then incubated at 37oC for indicated durations. Post-infection, samples were washed with HBSS, and fixed using 3.7% paraformaldehyde. Cells were stained for CEACAM, actin and bacteria and observed as described previously [88]. Intracellular bacteria were differentiated from extracellular bacteria via exclusion of antibody prior to membrane solubilization, as described [204].

3.3.7 Oxidative Burst Assays

For chemiluminescence-based oxidative burst assays, 5x105 cells were incubated with 25 µg/ml 5-amino-2,3-dihydro-1,4-phthalazinedione (‘luminol’; Sigma) in a volume of 100 µl, and then treated with agonists in a total volume of 200 µl, in triplicate. Infections proceeded for

60 min at 37oC, after which luminescence was read using a Tecan plate reader with i-control software.

3.3.8 Cytokine Measurements

5x10^5 neutrophils were infected with N. gonorrhoeae at MOI of 10 and incubated at 37oC for 3 h. At the experimental endpoint, the cells were pelleted by centrifugation at 2400 g for 5 min at 4oC, and supernatants were collected. Quantitative measurements of MIP-1α and MIP-2 chemokines was then performed using ELISA kits from R&D Systems.

63 3.4 RESULTS

3.4.1 CEACAM3 acts in concert with TLR4 to amplify cytokine secretion

N. gonorrhoeae does not naturally colonize the mouse urogenital tract, due in part to the lack of human CEACAMs to which the bacteria adhere within the mucosal epithelia; while mice do express CEACAMs, they are not recognized by the gonococcal Opa protein adhesins [242]. The simple expression of cDNA alleles expressing either human CEACAM1, CEACAM3 or CEACAM6 allows mouse neutrophils to engulf Opa+ N. gonorrhoeae, however CEACAM3 is necessary and sufficient for these cells to take up the bacteria via a process that involves assembly of actin-based phagocytic cups and a degranulation response reminiscent of human neutrophils [212]. Using the CEABAC transgenic mice, which contain a human genomic fragment encoding several different human CEACAMs, I have previously shown that the full length human CEACAM3 protein is expressed in mouse neutrophils in a manner reminiscent of that seen in humans, and that this allows the neutrophils to undergo an effective Opa-dependent phagocytosis of N. gonorrhoeae, degranulation, and inflammatory cytokine response to infection [258]. To simplify analysis of how CEACAM3 elicits the NF-κB-dependent transcriptional response, I herein sought to use ligation of CEACAM3 with specific antibodies. Intriguingly, while CEACAM3 is able to facilitate phagocytosis of even large (5.6 µm) anti-CEACAM- coated particles [195], crosslinking of CEACAMs on the neutrophil surface failed to activate an inflammatory response in either human or mouse PMNs (Fig. 3-1A, B). Since past work had looked at cytokine responses to whole bacteria, this prompted me to consider that CEACAM3 might act in collaboration with other innate pattern recognition receptors [8-11] to mount an inflammatory response. Interesting in this regard, despite the fact that PMNs express a broad variety of TLRs [104], vigorous inflammatory response to N. gonorrhoeae does not occur when the neutrophils lack CEACAM3 and/or the bacteria lack Opa [258], which would imply that the response may be co-dependent. To investigate the potential collaboration between CEACAM3 and other innate receptors in human peripheral blood PMNs, I treated hPMNs with LPS in combination with CEACAM- specific or control antibodies. Strikingly, a synergistic increase in IL-8 production was apparent when LPS was combined with anti-CEACAM, but not control antibody, in all donors tested (Fig. 3-1A). This synergy was also apparent when CEABAC PMNs were treated with LPS and

Figure 3-1. CEACAM cross-linking enhances LPS-induced cytokine production by hPMNs. (A) hPMNs were stimulated with anti-CEACAM antibody or control IgG, with or without LPS, as indicated. IL-8 production was measured 4 h post-infection. Each symbol represents a different donor. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (B) CEABAC PMNs were stimulated with anti-CEACAM antibody or control IgG, with or without LPS. MIP-2 production was measured 24 h post-infection. Error bars indicate SE, N=3 (WT: N=1).

65 anti-CEACAM antibody, as shown by dramatic increase in secretion of MIP-2, a functional homologue and a murine counterpart of human IL-8 [261]. This increase was not apparent when no antibody, or control immunoglobulin was used in place of anti-CEACAM antibody (Fig. 3- 1B). In both human and mice, the response to the combination of agonists was markedly greater than that seen in response to either alone, suggesting a synergy between the TLR4 and CEACAM3 receptors. The lack of response to antibody cross-linking suggests that CEACAM3-mediated phagocytic and inflammatory pathways are functionally distinct. This model is supported by data presented in the previous chapter, where I was able to block inflammatory cytokine production without affecting bacterial phagocytosis (Fig 2-6), however the molecular events that allow separation of these two different CEACAM3 effector functions are not known. Moreover, the molecular mechanism that allows synergy between ITAM-containing phagocytic receptors and TLRs remains poorly defined [262].

3.4.2 Rac2-deficient neutrophils readily engulf N. gonorrhoeae, but fail to mount oxidative burst and inflammatory responses

While mutant alleles lacking the cytoplasmic domain of CEACAM3 can mediate reduced but still significant engulfment of N. gonorrhoeae by transfected epithelial cells [204,207] and neutrophil cell lines [212], ITAM phosphorylation is required for Syk and PI3K activation, as well as the actin-dependent phagocytosis of N. gonorrhoeae or large (5.6 µm) CEACAM-specific antibody-coated particles [195,197,204,205,208,212]. The expression of dominant-negative Rho family GTPases in CEACAM3-transfected 293T cells or human neutrophils suggest that Rac facilitates this process [196,208]. Notably, Rac appears to be activated through both Syk-dependent [195,212] and Syk-independent [197,202] pathways, both of which are dependent upon phosphorylation of the CEACAM3 ITAM. In this context, it is pertinent to consider that the inhibition of CEACAM3 signaling blocks the phagocytic uptake of Opa protein-expressing N. gonorrhoeae by neutrophils, suggesting that CEACAM3 is the principle phagocytic effector for the gonococci despite the presence of CEACAM1, CEACAM6, and other potential receptors expressed by these cells [194]. In contrast to epithelial cells and fibroblasts, neutrophils express two different Rac isotypes, Rac1 and Rac2 [263]. Despite their high degree of homology, Rac1 and Rac2 have significant functional distinctions. While Rac1 is necessary to orient PMNs towards chemoattractants, Rac2 (but not Rac1) deficiency results in broad defects in chemotaxis,

66 opsonin-dependent phagocytosis, exocytosis of primary granules, and production of reactive oxygen species [207,236,238,264-266]. Thus, I considered how Rac2 contributed to CEACAM3-dependent functions, including the inflammatory response. To address this, CEABAC mice were bred with a Rac2-deficient mouse line (Rac2-/-). As reported previously, only CEABAC and not WT PMNs effectively bind and phagocytose Opa+ gonococci, with approximately 60% of bacteria internalized 30 min post infection (Fig. 3-2A). Unexpectedly, I did not observe a marked defect in phagocytosis of Opa+ N. gonorrhoeae in Rac2-deficient CEABAC PMNs (Fig. 3-2A), indicating that N. gonorrhoeae uptake by neutrophils can occur without Rac2. Interesting in this regard, prior studies demonstrate that the deletion of the CEACAM3 ITAM or loss of Rac function causes a reduction in N. gonorrhoeae uptake rather than a complete loss in the ability to engulf these bacteria in both model (CEACAM3- transfected) phagocytes and in neutrophils [196,204,208]. This suggests that an alternate pathway for bacterial engulfment may be engaged in the absence of normal CEACAM3 signaling. Opa+ N. gonorrhoeae stimulate production of reactive oxygen species in human PMNs, and this response is dependent of Src family kinases and PI3K [195,212,213]. Previous work from our lab has shown that wild type mouse PMNs do not produce ROS when infected with N. gonorrhoeae, but neutrophils from CEABAC mice respond in a manner similar to that seen with hPMNs due to Opa protein-dependent CEACAM3 binding. Since Rac2-deficient PMNs fail to mount a robust oxidative burst when treated with phorbol myristate acetate (PMA) [267], I sought to test whether Rac2 is involved in CEACAM3-induced oxidative burst response despite their normal gonococcal engulfment. Relative to CEABAC-derived neutrophils, the CEABAC Rac2-/- cells had a significant defect in ROS production (Fig. 3-2B), indicating a defect irrespective of the normal bacterial uptake. Given that the neutrophil inflammatory response to N. gonorrhoeae is also CEACAM3- dependent [258], I next compared MIP-1α and MIP-2 secretion in CEABAC and Rac2-/- CEABAC PMNs infected with N. gonorrhoeae. A marked reduction in the production of both chemokines was apparent with the Rac2-deficient cells, revealing a previously uncharacterized role for Rac2 in the inflammatory responses to N. gonorrhoeae (Fig. 3-2C).

68 Figure 3-2. Rac2-deficient CEABAC PMNs readily phagocytose N. gonorrhoeae, but fail to mount oxidative burst and inflammatory responses. (A) N. gonorrhoeae (MOI 10) binding and engulfment were compared in PMNs from CEABAC or Rac2-deficient CEABAC mice, 30 min post-infection. Intracellular and total PMN-associated bacteria were differentially stained, and quantified via immunofluorescence microscopy, N=2. (B) Oxidative burst by wild type, CEABAC and Rac2-deficient CEABAC mouse-derived neutrophils at 30 min post-infection with N. gonorrhoeae (MOI 25), N=2. Oxidative burst in response to PMA was also measured (N=1) (C) MIP-1α and MIP-2 chemokines in supernatants collected 3 h after PMN infection with N. gonorrhoeae (MOI=10), N≥2.

69 3.4.3 Bcl10-deficient neutrophils fail to respond to N. gonorrhoeae infection

B Cell Lymphoma/Leukemia 10 (Bcl10) protein has been shown to act upstream of Rac GTPase so as to mediate actin rearrangements in T cells and phagocytosis in macrophages [268,269]. Independent of its function in actin dynamics, Bcl10 is also an essential part of a protein complex (consisting of CARD-domain containing adaptor protein, as well as the paracaspase MALT1) responsible for NF-κB activation in both lymphocytes and myeloid cells [270]. Consequently, Bcl10 is essential for cytokine production downstream of a number of ITAM receptors in both macrophages and DCs [271]. Consistent with its central role in these various pathways, Bcl10 deficiency in humans is associated with severe immunodeficiency stemming from defects in both myeloid and lymphoid cells [272]. Relevant to this study, there is some indication that Bcl10 might also be involved in TLR signaling, although the results vary and seem to be both cell type- and context-dependent. In B cells, Bcl10 deficiency results in defects in cytokine production downstream of TLR4 signaling [273], TLR4 in gut epithelial cells seem to signal through both Bcl10-dependent and Bcl10- independent pathways [274], while findings in macrophages and DCs are not consistent, with some studies reporting a defect in TLR4 signaling [275,276] while others do not [181]. Considering that the role of Bcl10 in neutrophils has not been investigated, and taking into account the variability in responses between different cell types, I sought to test whether Bcl10 was involved in phagocytic and/or inflammatory signaling downstream of CEACAM3. I first examined uptake of Opa+ N. gonorrhoeae in WT or CEABAC PMNs with and without Bcl10, and found that loss in Bcl10 expression had no effect on bacterial internalization by the neutrophils (Fig. 3-3A). However, in contrast to the defect apparent in Rac-deficient PMNs (Fig. 3-2B), the oxidative burst response elicited by CEACAM3 engagement was not different in PMNs that lack Bcl10 (Fig. 3-3B), indicating that Rac2 and Bcl10 have distinct roles downstream of CEACAM3. In macrophages and DCs, loss of Bcl10 abrogates cytokine production otherwise triggered upon engagement of ITAM receptors, such as Dectin-1, suggesting a downstream decoupling of normal ITAM-dependent responses [276,277]. This prompted me to test whether the loss of Bcl10 affected the CEACAM3-dependent neutrophil inflammatory response to N. gonorrhoeae. While CEABAC PMNs elicit a robust MIP-1α and MIP-2 in response to Opa-

71 Figure 3-3. Bcl10-deficient neutrophils fail to respond to N. gonorrhoeae infection. (A) Bacterial binding and internalization were evaluated in PMNs from wild type, CEABAC and Bcl10-deficient CEABAC mice 30 min after infection with N. gonorrhoeae (MOI 10). Intracellular and total PMN-associated bacteria were differentially stained using gonococcal- specific antibodies, and quantified via immunofluorescence microscopy. N=2. (B) Reactive oxygen production by PMNs 30 min post-infection (MOI 25). N=2. (C) Quantification of MIP- 1α and MIP-2 chemokines from primary mouse PMN culture supernatants 3 h post-infection (MOI 10), N=2.

72 expressing N. gonorrhoeae, this response was absent in Bcl10-/- PMNs (Fig. 3-3C). This implicates Bcl10 as an essential mediator of CEACAM3-driven cytokine production, and reveals that Bcl10 couples ITAM activation to the NF-κB dependent inflammatory response seen in N. gonorrhoeae-infected neutrophils, analogous to what has been observed in other myeloid cells and lymphocytes.

3.4.4 Malt1-deficient neutrophils mirror the response seen with Bcl10 deficient PMNs

MALT1 is a crucial component of various signaling pathways involved in both innate and adaptive immunity, and functions with Bcl10 as a positive regulator of antigen receptor signaling [271]. However, while the defects seen in T cell activation of Bcl10-deficient mice reflect those seen with MALT1 deficiency, MALT1, unlike Bcl10, is largely dispensable for B cell activation and proliferation upon BCR stimulation [260]. Together, these observations suggest that the contribution of MALT1 depends on cellular context and/or are pathway- specific. In the context of innate immune signaling, MALT1 does contribute to the fungal cell wall (zymosan)-specific Dectin-1 receptor-dependent signaling in DCs, where it forms a complex with CARD9 and Bcl10 that leads to activation of NF-κB, but does not seem to contribute to LPS-dependent responses [181]. In accordance with the defect in cytokine production I observed in Bcl10-/- CEABAC PMNs, MIP-1α and MIP-2 secretion was also impaired in MALT1-/- CEABAC PMNs, while their phagocytosis and ROS responses were not affected (Fig. 3-4A-C). This is consistent with the effect of MALT1 and Bcl10-containing complexes contributing to the Dectin-1 response [181], and is the first indication that Bcl10 and MALT1 function together in neutrophils.

3.4.5 Bcl10 and MALT1 deficiency shows selective defect in TLR signaling in CEABAC PMNs

In considering that Bcl10 and MALT1 are essential for cytokine production by CEABAC PMNs, and of their contribution to other pathways, I considered whether they could represent a point of convergence between CEACAM3 and other classes of innate receptor. While PMNs have been shown to express all TLRs other than TLR3 [278], the molecular mediators involved have been rarely considered in this cell type. Moreover, contribution of the Bcl10-MALT1 complex to TLR signaling in general remains unclear [271]. The PRRs TLR2, TLR4 and TLR9 are triggered during N. gonorrhoeae infection [8-11]. Thus, to establish

74 Figure 3-4. Contribution of MALT1 to the PMN response to N. gonorrhoeae. (A) Bacterial binding and phagocytosis were evaluated in wild type, CEABAC or CEABAC Malt1-/- PMNs 30 min post-infection with N. gonorrhoeae. Intracellular and total PMN-associated bacteria were differentially stained, and quantified via immunofluorescence microscopy (MOI10) N=1. (B) Oxidative burst 30 min post infection (MOI 25). Representative of N=2 (N=2 for PMA response) (C) MIP-1α and MIP-2 chemokine in culture supernatants from N. gonorrhoeae- infected PMNs 3 h post-infection (MOI 10), N=1.

75 whether these responses could be effected by Bcl10 and/or MALT1, PMNs from the wild type or mutant backgrounds were stimulated with synthetic triacylated lipopeptide (Pam3CSK4), LPS, and synthetic oligonucleotides containing unmethylated CpG dinucleotides (CpG) as prototypical agonists that activate TLR2, TLR4 and TLR9, respectively. Interestingly, there was a selective defect in cytokine production downstream of TLR2 and TLR9 in both Bcl10 and MALT1-deficient CEABAC PMNs, while TLR4 signaling seems to be independent of both effectors (Fig. 3-5A,B). Additionally, it appears that cytokine responses triggered by different ligands may vary depending on the receptor triggered, since TLR4 and TLR2 led to robust induction of MIP-1α and MIP-2, while MIP-1α but not MIP-2 secretion was detected in PMNs treated with CpG (Fig. 3-5A,B). In summary, there was a reduction in TLR-dependent cytokine secretion in the absence of either Bcl10 or MALT1, suggesting a role for this complex downstream of TLR receptors in neutrophils.

3.4.6 Bcl10 regulates the synergistic cytokine production elicited by CEACAM3 and TLR4

Considering that Bcl10 and MALT1 are essential for CEACAM3-dependent inflammatory response and certain, but not all, TLR signalling, I considered that I could take advantage of the normal endotoxin response to explore the synergistic relationship between the CEACAM3 and TLR4 signalling pathways. To this end, PMNs from CEABAC, CEABAC Bcl10-/- or CEABAC MALT1-/- backgrounds were treated with various combinations of CEACAM-specific antibodies and endotoxin. While I observed a reduction in N. gonorrhoeae induced MIP-2 production in cells lacking Bcl10 (Fig 3-3C), the synergistic response between CEACAM3 and TLR4 was diminished, but not absent in Bcl10 deficient PMNs (Fig 3-5B). Moreover, MALT1 deficiency had no effect on MIP-2 production in response to LPS and/or CEACAM cross-linking (Fig 3-5D). This was unexpected given that it suggests that Bcl10 and MALT1 have distinct contributions to the synergy response, whereas previous findings by myself (Figs. 3-3, 3-4 and 3-5A,B) and others [270] were consistent with them functioning together.

Figure 3-5. Contribution of Bcl10 and MALT1 to the synergistic CEACAM3 and endotoxin response. (A-B) MIP-1α and MIP-2 responses to TLR ligands were measured in (A) Bcl10- and (B) MALT1-deficient backgrounds using specific agonists. Cytokine production was measured 24 h after stimulation. (Ai, ii, iii N=3; Bi, ii N=2, Biii N=1)(C-D) CEABAC PMNs deficient in either Bcl10 (C) or MALT1 (D) were treated as described for figure 3-1, and MIP-2 secretion was analyzed 24 h after stimulation. (C) N=3, (D) N=2.

77 3.5 DISCUSSION

The rapid and efficient detection and appraisal of incoming threats is a critical feature of the innate immune response. While detection largely relies on families of PRRs that recognize molecular patterns common to a wide range of pathogens, there is an emerging appreciation that this response is facilitated by more specific innate immune receptors that recognize a narrower range of ligands limited to specific sets of pathogens. Further specificity can be achieved by collaborative interaction between different PPRs, where simultaneous activation of multiple signals allows tailoring of the initial inflammatory response depending on microbial threat, and modulation of future adaptive responses. The neutrophil-restricted receptor CEACAM3 is decoy receptor engaged by pathogens with CEACAM-specific adhesins, a group that includes N. gonorrhoeae, N. meningitidis, H. influenzae and M. catarrhalis. Originally described as the main receptor responsible for phagocytosis and killing of CEACAM-binding N. gonorrhoeae [196], CEACAM3 has emerged as a dual function receptor, also responsible for the induction of a rapid and intense inflammatory response by activating cytokine secretion by PMNs [258]. At first glance, coupling of antimicrobial and inflammatory functions seems an obvious evolutionary benefit, allowing direct restriction of bacterial spread while mobilizing other immune cells to help. However, as evident from the immunopathogenesis associated with gonorrhea, it also creates the dangerous potential to drive an overzealous and ultimately detrimental immune response. Limiting the extent of CEACAM3-driven inflammation might therefore be an effective approach to limit the immunopathology. I have previously reported that N. gonorrhoeae binding to CEACAM3 elicits a pro- inflammatory transcriptional program that drives the neutrophil cytokine response [258]. Herein, I reveal that CEACAM3 cannot act alone in this regard, but instead acts in synergy with TLR4 in both human and CEABAC neutrophils. This effect is manifested as an induction of chemokine response that is greater than the sum of that seen to either CEACAM3 ligation or endotoxin alone. Bcl10 and MALT1 are both required for the CEACAM3-dependent inflammatory response to N. gonorrhoeae infection, but are not required for neutrophils to respond to purified endotoxin. Interesting in this regard, the chemokine response elicited upon direct ligation of CEACAM3 in the presence of endotoxin is normal in MALT1-deficient mice, making it enticing to consider that this effector facilitates CEACAM3 signalling but is not necessary for the synergy between CEACAM3 ITAM and TLR4-dependeng signals.

78 Additionally, I also demonstrate the involvement of both Bcl10 and MALT1 in TLR2 and TLR9, but not TLR4 signalling. The fact that the defects in TLR2 and TLR9 were only partial could originate in evolution of ‘backup’ alternative signalling pathways, or could indicate that Bcl10-MALT1 function to increase the efficacy of the response. Considering that the effect depends upon the innate immune agonist, further studies are needed to define the specific contribution of Bcl10 and MALT1 in signalling by each TLR. The fact that Bcl10 deficiency suppresses the synergy between TLR4 and CEACAM3 suggests that Bcl10 plays some function distinct from the MALT1-Bcl10 complex, or that it is more critical for the activity of this complex. Another notable outcome of our experiments is the divergence between MIP-1α and MIP-2 responses, since the effect of MALT1 and Bcl10 deficiency tended to be more pronounced on the latter chemokine, and the synergistic response was specific to MIP-2. This highlights that it is not an ‘all or none’ response and illustrates how the integration of receptor signalling can fine-tune the resulting inflammation. While my findings represent the first indication of a function for Bcl10 and MALT1 in neutrophils, these effectors form a complex with CARD9 to transduce signals from ITAM- associated receptors in DCs and macrophage [181,276]. When considered in the context of my previous findings that Syk and Tak1 are required for CEACAM3-dependent NF-κB transcription (Chapter 2), it seems likely that the CEACAM3 engages the same Syk/CARD9- Bcl10-MALT1/Tak1 signalling axis as does the fungal-specific C-type lectin receptor, Dectin-1 [173]. Interesting in this regard, it is important to recall that that inhibition of CEACAM3- mediated phagocytosis does not result in inhibition of inflammatory signalling, and vice versa [258]. This suggests that the two pathways can proceed independently of one another and CEACAM3 inflammatory function can be controlled without affecting PMNs ability to mount bactericidal responses to N. gonorrhoeae infection. The discovery and characterization of receptors specializing on microbe detection have revolutionized our understanding of innate immunity. More recently, uncovering of dual function receptors, such as CEACAM3 and the fungal receptor Dectin-1, which function in both detection and direct phagocytosis of pathogens, brings another level of complexity to this field. These receptors provide new paradigms by which to understand the molecular networks that connect PRR with other microbial-derived cues that allow the host to respond to a microbial threat in a rapid and precise manner.

79 4 Selection for CEACAM receptor-specific binding phenotype during Neisseria gonorrhoeae infection of

the human genital tract

This Chapter has been published as: Sintsova A*, Wong H*, MacDonald K, Virji M, Kaul R, Gray-Owen SD. Analysis of clinical Neisseria gonorrhoeae isolates reveals selective binding to different CEACAM family receptors. Infect Immun. 2015 Apr; 83(4):1372-83.

*Equal contribution

Acknowledgements: Dr. Wong contributed to Figure 4-2, 4-3, 4-4A, and Table 4-1.

4.1 ABSTRACT

Infections by Neisseria gonorrhoeae are increasingly common, are often antibiotic resistant, and can result in serious and lasting sequelae, prompting gonococcal disease to re- emerge as a foremost global health concern. N. gonorrhoeae is a human-restricted pathogen that primarily colonizes urogenital mucosal surfaces. Disease progression varies greatly between sexes: men usually present with symptomatic infection characterized by a painful purulent urethral discharge, while in women the infection is often asymptomatic, with most severe pathology occurring when the bacteria ascend from the lower genital tract into the uterus and fallopian tubes. Classical clinical studies demonstrated that clinically infectious strains uniformly express Opa adhesins, however the specificities of these were unknown at the time. While in vitro studies have since identified CEACAM proteins as the primary target of Opa proteins, the gonococcal specificity for this human family of receptors has not been addressed in the context of natural infection. In this study, Dr. Wong and I characterize a collection of low- passage clinical specimen-derived N. gonorrhoeae for Opa expression and assess their CEACAM binding profile. We report a marked in vivo selection for expression of phase variable Opa proteins that bind CEACAM1 and CEACAM5, but selection against expression of Opa variants that bind to the neutrophil-restricted decoy receptor CEACAM3. This is the first study showing phenotypic selection for distinct CEACAM-binding phenotypes in vivo, and supports the opposing function of CEACAMs that facilitate infection versus drive inflammation within the genital tract.

4.2 INTRODUCTION

Neisseria gonorrhoeae has persisted in the human population despite all attempts to limit the spread of infection [2]. The alarming rise in antibiotic-resistant strains and increase in global incidence of infection have put N. gonorrhoeae at the forefront of national and international public health agendas [1]. N. gonorrhoeae is a sexually transmitted pathogen that most commonly colonizes the urogenital mucosa, although it may also be found on nasopharyngeal, rectal and ocular surfaces. Disease manifestation varies greatly between men and women. Infections in men are commonly characterized by acute urethritis with profuse purulent discharge. This pus largely consists of polymorphonuclear leukocytes (PMNs), potently phagocytic cells responsible for bacterial elimination, but with the potential to damage the surrounding tissues in the process [7]. In women, N. gonorrhoeae colonizes the endocervix, where it also has the potential to cause painful inflammation and cervical discharge; however, most infections in women are asymptomatic [1]. If left untreated, the gonococci may ascend into the female upper genital tract to promote a pathogenic inflammatory response that can precipitate severe health issues, including pelvic inflammatory disease (PID), ectopic pregnancies and infertility [7]. N. gonorrhoeae is a human-restricted pathogen that has evolved sophisticated mechanisms to facilitate colonization and persistence within its host. Essential for these processes are specialized adhesins that allow N. gonorrhoeae attachment to receptors expressed exclusively on human mucosal tissues. The type IV pilus mediates the initial bacterial attachment to the host cell. By virtue of its ability to retract, the pilus overcomes mucosal flow and brings the bacteria into close proximity with the epithelial cell [17] so as to facilitate a more intimate association and/or cellular invasion [279]. Studies using male human volunteers indicate that pilus is not required for initial infection, though may contribute to disease manifestations [20]. The colony-opacity associated (Opa) proteins were recognized by their effects on inter- bacterial aggregation and leukocyte association [219,280,281], and later shown to mediate a tight secondary association between N. gonorrhoeae and the epithelia [235]. Each gonococcal isolate possesses ~11 different opa genes, each encoding antigenically and phenotypically distinct variants that reversibly turn expression ‘off’ and ‘on’ at a rate estimated to be 10-3-10- 4/cell/generation [23,24]. N. gonorrhoeae isolated from both naturally infected men and women are predominantly Opa+, as are isolates obtained from men experimentally infected with

82 transparent (Opa phase-varied ‘off’) colonies [218-220]. Most gonococcal Opa variants bind to one or more members of the human CEACAM family of receptors [81,85-88,222,282]. CEACAM receptors are members of the immunoglobulin (Ig) superfamily, containing an Ig variable region-like N-terminal domain followed by a varying number of Ig-constant region-like domains exposed at the cell surface [34,283]. CEACAM1, CEACAM3, CEACAM5 (CEA) and CEACAM6 (but apparently not other CEACAMs) are each capable of mediating neisserial adherence to and engulfment by the various tissues on which they are differentially expressed [81,85-88,223,284). Epithelial CEACAMs (CEACAM1, CEACAM5, and CEACAM6] are all presumed to facilitate bacterial colonization [30,226]. In the female genital tract, squamous epithelia express CEACAM5, whereas CEACAM1 is expressed on columnar epithelia of the endocervix and uterus [36], allowing each to be accessible for direct docking by the gonococci. Moreover, CEACAM1 is widely expressed on lymphocytes and CEACAM1-induced signalling can influence immune cell activation [45,51,89,90,92,93,227,285], potentially providing a mechanism for immune evasion by N. gonorrhoeae. While no study to date has looked at CEACAM expression within the male urethra, a transgenic mouse line expressing human CEACAMs in the manner that closely reflects the spatiotemporal expression pattern in humans expresses CEACAM5 on the urethral mucosal surface [234]. Despite an abundance of evidence for importance of Opa-CEACAM interaction for N. gonorrhoeae infection in vitro, the contribution of this association is challenging to assess in vivo. Most characterized gonococcal isolates bind to human CEACAM1 [282], but their ability to bind other CEACAMs remains uncertain. Due to host specificity of Neisseria, mouse models remain limited. However, expression of human CEACAM1 in a mouse allowed persistent colonization by N. meningitidis [201], and a mouse model expressing human CEACAM5 showed increased gonococcal recovery from lower genital tract [200]. Contrastingly, Opa binding to neutrophil CEACAM3 drives inflammation and gonococcal clearance [195,196,202,204,212-214,228,258]. The specific contribution of Opa binding to individual CEACAMs for N. gonorrhoeae colonization and pathogenesis in humans, and how differences in CEACAM distribution between the sexes might affect the outcome of infection, has not been addressed. In this study we sought to characterize gonococcal CEACAM binding phenotypes expressed within the human urogenital tract. To this end, we obtained a collection of primary low-passage clinical isolates (LPCIs) of N. gonorrhoeae from male urethral swabs or endocervical specimens taken from a sexually transmitted disease clinic [286]. Since binding

83 specificity cannot be predicted by the antigenically hypervariable Opa protein sequences, a post doctoral fellow in our lab, Dr. Wong, developed a high-throughput binding assay with transfected cells expressing individual human CEACAMs to define the specificity of each colony phenotype apparent within each low-passage clinical specimen. He observed a clear selection for expression of Opa variants that bind to epithelial and leukocyte-expressed CEACAMs with concomitant selection against a CEACAM3-binding phenotype. I also show that consistent with the detrimental effects of CEACAM3-dependent binding by neutrophils, a phase variant that binds to CEACAMs was actively phagocytosed, activated production of reactive oxygen species, promoted degranulation, and activated the release of pro-inflammatory cytokines from neutrophils, while its Opa-deficient variant did not. Together, these findings support a model in which Opa protein phase variation allows in vivo selection for neisserial phenotypes that facilitate persistence within the mucosal tissues.

84 4.3 MATERIALS AND METHODS

4.3.1 Reagents

Lipofectamine and hygromycin B were obtained from Invitrogen (Burlington, Ontario, Canada). Heparin sodium salt, β-mercaptoethanol (BME), paraformaldehyde (PFA), gelatin and p-nitrophenyl phosphate (pNPP) were obtained from Sigma (Oakville, Ontario, Canada). Fraction V bovine albumin (BSA), 5-bromo-4-chloro-3-indoyl phosphate disodium salt (BCIP), nitrotetrazolium blue chloride (NBT) and G418-sulphate were obtained from Bioshop (Burlington, Ontario, Canada). Monoclonal antibody 4B12/C11, which recognizes all gonococcal Opa variants described, was generously provided by Dr. M. Achtman (Max-Planck- Institut für Infektionsbiologie) [287]. The monoclonal antibody against gonococcal pilus is a kind gift from Dr. M. So (Oregon Health Sciences University) [288]. Anti-gonococcal polyclonal antibody UTR01 was generated by three subcutaneous immunizations with killed N. gonorrhoeae N302 (Opa–) as previously described [204]. Alkaline phosphatase (AP)-conjugated goat-anti-rabbit IgG(H+L), horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG(H+L) and AP-conjugated goat-anti-human IgG(H+L) antibodies were purchased from Jackson ImmunoResearch Laboratories (Mississauga, Ontario, Canada). The soluble CEACAM1-Fc fusion protein was prepared as described [223]. The CEACAM-specific- phycoerythrin conjugate (CD66-PE; Clone B1.1) was obtained from Becton Dickinson. MICORTEST 96 tissue culture plates were obtained from Becton Dickinson Labware (New Jersey, USA). 4.3.2 Bacterial strains and growth conditions

Low-passage clinical isolates (LPCIs) of N. gonorrhoeae were cultured from urethral and endocervical swabs in a sexually transmitted disease clinic or as part of a longitudinal study of 302 commercial sex workers in the lower socioeconomic district of Pumwani in Nairobi, Kenya, from July 1991 to December 1995 [286]. All male patients were symptomatic at the time of culture isolation. Swabs were streaked onto Thayer-Martin medium and incubated at 37°C and 5% CO2 for 48 h before further characterization. N. gonorrhoeae was identified by colony morphology, oxidase test, and Gram stain reaction. Specimens were subcultured once after multiple colonies were picked, resuspended in skim milk containing 10% glycerol, and frozen at -70°C. Colony phenotypes were routinely monitored using a binocular microscope. Each colony phenotype representing more than 5% of colonies from these primary stocks were selected for

analysis. Isogenic N. gonorrhoeae MS11-derived strains N302 (Opaˉ), N303 (Opa50, which binds HSPG-specific receptors), N309 (Opa52, which binds CEACAMs 1,3,5, and 6), N311

(Opa54, which binds CEACAMs 1 and 5), and N313 (Opa57, which binds CEACAMs 1,3,5, and 6), and N496 (Opaˉ pilus+) were described previously [81,235] and were generously provided by Dr. T. F. Meyer (Max-Planck-Institut für Infektionsbiologie, Berlin, Germany). Apart from strain N496, these derivatives of N. gonorrhoeae strain MS11 are pilus deficient and contain a chromosomal deletion of the opaC30 locus encoding the only Opa protein variant that mediates HSPG-dependent host cellular invasion by this strain [235]. Due to the phase-variable expression of pilus and Opa proteins, the gonococcal strains were cultured from frozen onto GC agar 2 days prior to experimentation, colonies with the desired Opa and pilus phenotypes were visually identified with a binocular microscope and then sub-cultured to provide bacteria for experimentation on the subsequent day. Opa expression of gonococcal isolates used for infection experiments was confirmed by immunoblot analyses of total bacterial lysates using monoclonal antibody 4B12/C11 which recognizes all Opa protein variants described [287], and pilus expression was monitored using 10H5.1.1 antibody, which recognizes conserved SM1 epitope [288].

4.3.3 Cell lines

All cell lines were grown in RPMI 1640 containing L-glutamine and 5% FBS

(Invitrogen) in a 37°C humidified incubator with 5% CO2. Lec11 cells, a CHO cell derivative [289], were used for expression of CEACAM receptors. CEACAM1 [193], CEACAM3 [208], CEACAM5, CEACAM6, CEACAM8 [290], and the expression vector alone were transfected into Lec11 cells by Lipofectamine according to manufacturer’s protocol. Transfected cell lines were maintained in medium containing 400 µg/mL G418-sulphate (CEACAM1, 5, 6, 8, and vector alone) or 200 µg/mL Hygromycin B (CEACAM3). Ten million CEACAM transfected cells were stained with the carcinoembryonic antigen antibody (DAKO, Denmark), which binds CEACAM1, CEACAM3, CEACAM5 and CEACAM6 [81], followed by Bodipy FL-conjugated goat-anti-rabbit IgG(H+L) (Molecular Probes) for fluorescent activated cell sorting using a FACSCalibur flow cytometer (BD Biosciences, Mississauga, Ontario, Canada) in order to remove the cells that do not express CEACAMs.

86 4.3.4 CEACAM receptor expression

Flow cytometric analyses were carried out in order to confirm the surface localization of CEACAM receptors in Lec11 cells. Briefly, approximately one million trypsinized cells were incubated with 0.2 mL of PBS/Mg/Ca containing 3% FBS (PBS/Mg/Ca/FBS) and 10 µg/mL CEA DAKO for 1 h on ice. Unbound antibody was removed by washing the cell pellet with 3 changes of PBS/Mg/Ca/FBS. Cell pellets were then incubated with 0.2 mL of PBS/Mg/Ca/FBS containing 4 µg/mL Bodipy FL-conjugated goat-anti-rabbit IgG(H+L), and unbound antibody was removed as described. The stained cells were fixed with PBS containing 1% PFA and fluorescence measured with a FACSCalibur. In 96-well binding assays, Lec11 monolayers were mock infected with serum-free RPMI media containing 200 µg/mL heparin and 0.2% BSA (RPMI/B/H) alone and stained with CEA DAKO followed by AP-conjugated goat-anti-rabbit IgG. Substrate development was carried out as described for bacterial binding.

4.3.5 96-well binding assay

The gonococcal isolates were divided into groups for experimentation in order to ease processing. For each binding experiment, the MS11 gonococcal strains, with defined Opa phenotype, were included as experimental controls. Each gonococcal strain was tested in triplicate wells, and the experiment was repeated to ensure reproducibility. 96-well plates were coated with PBS/Mg/Ca containing 0.2% gelatin (0.22µm filtered) for 30 min at 37°C prior to being seeded with Lec11 cells at 80% confluency. The next morning, the serum present in growth medium was removed by washing with PBS/Mg/Ca 3 times. Cells were then incubated with RPMI/B/H at 37°C CO2 until infection with bacteria, with the soluble heparin being maintained throughout infection so as to prevent HSPG-dependent bacterial binding to the mammalian cells [88]. Gonococci were washed with PBS/Mg/Ca to remove outer membrane blebs, and then resuspended in RPMI/B/H for density measurements. The bacterial suspensions were applied to each cell line at a MOI of 50. To synchronize infection, bacteria were centrifuged onto the monolayers at 67 g using a tabletop centrifuge at room temperature for 5 min. Infection was carried out at 37°C CO2 for 2 h, at which point non-adherent bacteria were removed by washing the wells 3 times with PBS/Mg/Ca. Monolayers were then fixed with PBS/Mg/Ca containing 1% PFA. To detect the presence of bound gonococci, PFA was first removed by 3 washes of PBS, and the monolayers were permeabilized with PBS containing 0.4% Triton X-100 for 10 min, followed by 3 washes of PBS. PBS containing 0.2% BSA was

87 used to block the wells for 15 min prior to addition of antibody UTR01 at 1:800 dilution for 1 h. Unbound antibodies were removed by 3 washes of PBS, followed by the addition of AP- conjugated goat-anti-rabbit IgG at 1:10,000 for 1 h and a final 3 washes of PBS. pNPP was used to reveal bound bacteria. Absorbance at 405nm was recorded 3 and 20 hours post-pNPP addition using a Titertek Multiskan Plus plate reader. All staining steps were carried out at room temperature.

4.3.6 Whole Cell Dot Blot

Gonococcal cells were washed once with PBS containing 1 mM MgCl2, 0.5 mM CaCl2, 0.22 µm filtered (PBS/Mg/Ca). Half a million cells in 3 µL volume were applied to nitrocellulose membrane and air dried. The membrane was incubated in PBS containing 0.05% Tween-20 (PBS-T) and 5% skim milk powder overnight at 4°C. The next morning, the membrane was incubated with 0.5 µg/mL of CEACAM1-Fc in blocker for 2 hours at room temperature. The membrane was extensively washed prior to incubation with AP-conjugated goat-anti-human IgG antibody at a 1:5000 dilution. Excess antibody was removed by repeat washings in PBS-T. Alkaline phosphatase reaction was carried out by pre-equilibrating the membrane in 0.1 M Tris pH 9.5, and subsequently incubated in a solution containing 0.1 M Tris pH 9.5, 7 mM MgCl2, 135 µM BCIP and 122 µM NBT.

4.3.7 Statistical analysis

Bacterial strains were tested in groups with MS11 strains as controls. In order to present and compare all the data, binding data from the various plates were normalized to the MS11 controls. Data from two MS11 strains that fall within the dynamic range of the substrate reaction were chosen for the transformation of binding data from individual experiments. Briefly, the averages for the two MS11 control strains were first normalized to their respective cumulative means. A normalization factor for each experiment was calculated by taking the average of the two normalized MS11 averages. Data from each experiment are presented as the average between the triplicate wells, subtracting the averages from the ‘No GC’ control wells, and transformed according to the normalization factor. Standard deviation was calculated from the triplicate wells, and adjusted with the same normalization factor.

4.3.8 Primary Neutrophil Isolation

Human neutrophils were isolated from citrated whole blood taken from healthy

88 volunteers by venipuncture using Ficoll-Paque Plus (Amersham Biosciences; Buckinghamshire, England). Contaminating erythrocytes were removed by dextran sedimentation and hypotonic shock, as described previously [204].

4.3.9 Bacterial infections for immunofluorescence microscopy

5 5 x 10 PMNs were centrifuged onto coverslips at 514 g for 10 min. Cells were infected at MOI of 10 (for binding and internalization studies) in a volume of 500 µl, re-centrifuged for 5 min at 57 g to facilitate bacterial association with cells, and then incubated at 37oC for indicated durations. Post-infection, samples were washed with HBSS, and fixed using 3.7% paraformaldehyde. Cells were stained for CEACAM, and bacteria and observed as described previously [81]. Intracellular bacteria were differentiated from extracellular via exclusion of N. gonorrhoeae-specific antibody, as described [204].

4.3.10 Oxidative Burst and Degranulation Assays

For chemiluminescence-based oxidative burst assay, 5x105 cells were incubated with 25 µg/ml 5-amino-2,3-dihydro-1,4-phthalazinedione (‘luminol’; Sigma) in a volume of 100 µl, and then treated with agonists in a total volume of 200 µl, with each sample done in triplicate. Infections proceeded for 60 min at 37oC, after which luminescence was read using a Tecan plate reader with i-control software. For flow cytometry-based degranulation assay (CEACAM surface expression), 106 PMNs were treated with agonists in 500 µl of HBSS for 30 minutes at 37oC. Infections were stopped by centrifugation at 2,400 g for 3 min at RT. Cell pellets were fixed in 1% PFA, and stained with 1.25 µg of PE-conjugated rat anti-mouse CEACAM in a total volume of 200 µl. Elastase, and lactoferrin release assays were performed essentially as described by others [238]. Briefly, 106 PMNs were exposed to agonists in a total volume of 500 µl and then incubated for 1 h at 37oC. Cells were then pelleted and supernatants collected. For the elastase assay, 50 µl of supernatant was diluted 2-fold in PBS, incubated with 100 µl DQ elastin substrate conjugated to BODIPY FL (from the EnzCheck Elastase kit; Molecular Probes), and then incubated for 2 hours at RT before reading fluorescence with 488 nm excitation and 515 nm emission. For elastase assays, a percentage (%) release is shown, calculated as the amount of the protein in the supernatant divided by the amount released after PMA treatment. Lactoferrin release from PMN granules was assayed by ELISA as described by others [291]. For CEACAM release, 106 PMNs were infected for 1 h and fixed in 1% PFA. The cells were stained with CEACAM-PE and fluorescence was measured with FACSCalibur.

4.3.11 Cytokine Measurements

5x105 cells were infected with N. gonorrhoeae at MOI of 10 and incubated at 37oC for 3 h. Infections were then stopped by centrifugation at 2400 g for 5 min at 4oC, and supernatants were collected. Quantitative measurements of cytokines were performed using ELISA kits from BD Biosciences (IL-8, TNF).

90 4.4 RESULTS

4.4.1 Most low passage clinical isolates display Opa protein expression

Since the receptor specificity of Opa variants cannot be predicted from their protein sequence, we set out to assess the CEACAM binding phenotype of isolates minimally passaged from clinical specimens. We obtained stocks passaged twice (once from the specimen and once for freezing) from clinical samples taken from the male urethra or female endocervix. The 28 isolates selected represent the predominant gonococcal serovars circulating in Nairobi during the study period [286]. Western blot analysis using 4B12/C11 antibody that cross-reacts with all known N. gonorrhoeae Opa variants revealed that 96% of isolates expressed at least one Opa protein (27 out of 28 strains; Figure 1, Table 1). This is consistent with previous reports indicating that gonococcal strains isolated from natural infections express Opa proteins [218- 220]. We detected pilus in only 8 of 28 strains (29%; Table 4-1, Fig. 4-1). Since piliation is generally considered to be present in fresh clinical isolates, it is possible that strain piliation may have been lost by pilE recombination and/or pilC phase variation during subculture during in vitro subculture [292]. Microscopic examination of the LPCIs grown on GC agar revealed that each contained multiple distinct colony morphologies. Since gonococcal colony phenotypes often reveal changes in Opa and/or pilus expression, Dr. Wong considered each colony type separately. Sixty-four morphologically distinct gonococcal isolates were derived from the original 28 specimens, and each was then tested for Opa and pilus expression. Since phase variation is an ongoing process, sub-culturing was performed using a bottom-lit binocular dissecting microscope to confirm selection of colonies reflecting the originally-observed phenotype and exclude any visually-apparent phase variants arising during passage. Western blot analyses showed 64% (14 out of 22) male-derived isolates expressed detectable Opa variants, while 83% (35 out of 42) female-derived strains displayed one or more Opa variant (Table 4-1). Thus, while 96% of people in the cohort had Opa+ gonococci, and while the majority of colonies obtained from most specimens were Opa+, the infections often (6 of 18 female infections, 4 of 10 in males) also contained Opa- N. gonorrhoeae. While most isolates displayed a single Opa and/or pilus variant band on the immunoblots, it is pertinent to note that some of the isolates expressed more than one discernible Opa protein and/or pilin variants (Table 4-1). Indeed, since variants with similar electrophoretic mobility would not be discriminated, it is possible that this occurs more frequently than is apparent.

Table 4-1. Adhesin expression by a collection of low-passage clinical N. gonorrhoeae isolates. Summary of immunoblot analysis for Opa protein and pilin expression in low-passage clinical N. gonorrhoeae isolates. Colonies with distinct colony morphologies present within each original specimen were assigned an alphabetic designation, i.e. colony phenotype variants derived from original strain 4034 were labeled 4034A, 4034B, 4034C, and 4034D, and analyzed alongside the original strain. Numbers in Opa and Pilus columns indicate a number of distinct bands identified by Western blotting.

Figure 4-1. Opa protein expressing by LPCIs. Representative immunoblot with Opa-specific (4B12C11) and pilin-specific antibody (10H5.1.1) showing Opa and pilus expression in control - - + MS11 strains (Opa , Opa50, Opa52, Opa57, Opa pil ) and low-passage strains 4034 and 4058. 4034 A-D and 4058 A-C variants were derived from original specimens based on distinct colony morphology. Immunoblot results for all isolates are summarized in Table 4-1.

94 4.4.2 Establishing a high-throughput CEACAM binding assay

We hypothesized that the high prevalence of Opa expression in our LPCIs suggests that Opa proteins facilitate infection. However, since each Opa variant may have a different spectrum of CEACAM binding, since the spectrum of binding has the potential to drastically affect the host cellular response to infection [45,90,93,195,200,212,258], and since the pattern of CEACAM expression varies between sexes and cell types, Dr. Wong sought to characterize the binding specificity of each N. gonorrhoeae isolate. For this purpose, he transfected the Chinese hamster ovary-derived Lec11 cell line with individual CEACAM receptors (CEACAM1, CEACAM3, CEACAM5, CEACAM6, and CEACAM8). Since N. gonorrhoeae adhesins, such as pilus, have strict specificity for human receptors, this rodent cell line was chosen to eliminate any binding with endogenously- expressed cellular receptors. Expression and maintenance of human CEACAM receptors on the cell surface was confirmed by in situ staining (Fig. 4-2A) and flow cytometry (Fig. 4-2B). In order to verify that the CEACAM receptors expressed on Lec11 cells maintain their Opa binding specificity, and to validate our multiplexed approach, Dr. Wong tested the binding of well-defined Opa variants expressed by recombinant N. gonorrhoeae MS11 strains to these cell lines in a 96-well format binding assay. Since gonococci can express adhesins that bind HSPG receptors, including certain individual Opa variants [81,141,293], soluble heparin was maintained within the culture medium to block HSPG (but not CEACAM) binding [88]. The CEACAM-binding pattern observed for each strain tended to correlate with what was previously reported [81] (Fig. 4-2C). Wells that were mock infected (No GC) and gonococci that do not express Opa (Opa-) allowed us to establish background signals for N. gonorrhoeae and/or antibody binding to the Lec11 cell lines. Opa52-expressing gonococci bound CEACAM1-,

CEACAM3-, CEACAM5- and CEACAM6-expressing cells. While Opa57 is generally considered to also bind all four of these CEACAMs, its binding to CEACAM3 was modest in this assay system. As expected, Opa54-expressing gonococci only bound CEACAM1- and CEACAM5-expressing cells. Gonococci that express pili, but not Opa (Opa-Pil+) did not bind Lec11 cells, but instead showed evidence of binding to wells without Lec11 cells (‘Gelatin’ and

‘Medium’ wells). Opa50-expressing gonococci showed preference for Lec11 CEACAM1 cells and not any of the other cell lines. Although Opa50 interacts specifically with heparan sulfate proteoglycan (HSPG) receptors [28,88], it does display a low level of CEACAM1 binding [81].

Since Opa50 binds the carbohydrate component of HSPGs, the difference in binding could

96 Figure 4-2. Establishing 96-well CEACAM-binding Assay. (A) CEACAM expression by Lec11 cells. Lec11 cells transfected with the empty expression vector (V) or encoding individual CEACAM receptors were grown in gelatin-treated 96-well plates that also included control wells without cells but with gelatin treatment or medium alone. These were stained for CEACAM expression using CEA DAKO antibody and AP-conjugated goat-anti-rabbit secondary antibody, and then detected using a colorimetric reaction. The means of results from triplicate wells and their standard deviation are presented. (B) Flowcytometric analysis of CEACAM expression on the Lec11 cell surface. Lec11 cell lines were trypsinized and stained with DAKO CEA (black bar) or DAKO isotype control (white bar) antibodies, followed by BodipyFL-conjugated goat-anti-rabbit antibody. Lec11 cells were gated based on forward and side scatter profiles and the mean FL1 (BodipyFL) fluorescence is presented as histograms. (C) MS11 strains (MOI 50) were used to infect Lec11 cells in a 96-well binding experiment. 2 h post-infection, non-adherent bacteria were removed by washing, monolayers were fixed and permeabilized, and bound gonococci were detected using UTR01 antibody, followed by the goat anti-rabbit AP-conjugated secondary antibody. After developing with colorimetric reagent, averages of the absorbance at 405nm from triplicate wells with the standard deviation is presented.

97 plausibly be attributed to distinct CEACAM1 glycosylation patterns between the Lec11 cell lines and those previously used, however detailed analysis of CEACAM glycosylation in these versus other cells has not been performed.

4.4.3 LPCIs show distinct CEACAM-binding patterns

Next Dr. Wong used the high-throughput binding assay to determine the CEACAM- binding phenotypes of the gonococcal strains recovered from low-passage clinical samples. 82 of 92 isolates (89%) bind to one or more CEACAM. While most of these bind to both CEACAM1 and CEACAM5, ten strains are specific for CEACAM1 (2173B, 2174A, 2177A, 2177B, 2179A, 4002B, 4015, 4015A, 4121, 4174) and two are specific for CEACAM5 (2176, 2174B). Despite the high level of sequence identity between CEACAM1 and CEACAM3 (88% identical within the N-terminal domain that binds Opa), far fewer strains (27%) bound CEACAM3, and the level of binding to CEACAM3-expressing cells tended to be considerably less than that to the other CEACAMs (Fig. 4-3), suggesting that there was a selection against Opa variants that bind this neutrophil-restricted receptor. Among the original 28 LPCIs, CEACAM6 binding was more common in strains isolated from female (67%) than male (50%) infections (Fig. 4-4B). More strikingly, CEACAM5 binding was evident for all (100%) male isolates, but only for 67% of female-derived specimens (Fig. 4-4B). This suggests a stronger selection for CEACAM5-specific Opa variants in men versus women, though the relatively small sample size (10 male versus 18 females) makes it inappropriate to draw a definitive conclusion from this relationship. Finally, it is pertinent to note that, while none of the recombinant Opa variants tested to date are able to bind to CEACAM8 [81,86,87], several isolates showed very low level binding to CEACAM8-expressing cells (Fig. 4-3) whereas none bound to the untransfected Lec11 cell line (data not shown). Given that CEACAMs are heavily glycosylated, it is plausible that this interaction is distinct from the protein-protein-mediated binding between Opa and the other receptors [208]. However, since each of these isolates also bound to other human CEACAMs, it also seems feasible that certain variants have very low affinity for this closely related receptor. To validate the results of the Lec11-based infection assay, Dr. Wong used an established whole cell blot assay using soluble CEACAM1-Fc fusion protein [223] to reassess CEACAM1 binding specificity of the commonly used MS11 strains versus the LPCIs. As expected, recombinant MS11 strains expressing Opa57 N. gonorrhoeae showed binding to CEACAM1-Fc, - while Opa N. gonorrhoeae did not (Fig. 4-4A).

Figure 4-3. Low-passage clinical gonococcal isolates show distinct CEACAM-binding profiles. Binding data from 96-well binding experiments performed with Lec11 cells expressing indicated human CEACAMs. The grey bars represent the cumulative means of MS11 controls, black bars are the original low-passage clinical specimens, with their respective phenotypically- selected colony variants represented in adjacent white bars. The threshold for binding is set at one standard deviation above the MS11 control strain, and is represented as the dotted horizontal line. Asterisks indicate strains that were found to display significant levels of binding in two independent experiments. Percentages indicate the proportion of total (original isolates plus individual colony variants) shown to bind specific receptor.

99 The vast majority of the original (mixed phenotypes) LPCIs (91% of isolates) also bound to CEACAM1-Fc, corroborating the results from the 96-well assay, although certain variants displayed more clear binding to either the soluble (2174, 4001) or cell-expressed (4005) CEACAM1. Overall, these results suggest that there is a selective advantage for expressing Opa variants that bind CEACAM1 and CEACAM5 in vivo, presumably because these are expressed by mucosal epithelia. In contrast, the low level of binding to CEACAM3, which is a neutrophil- expressed phagocytic receptor, indicates that this may be detrimental to the bacteria.

4.4.4 CEACAM binding affects human neutrophil responses to LPCIs

Characterization of individual CEACAM function in neutrophilic cell lines showed that Opa-mediated neutrophil activation is mediated by CEACAM3 signalling, while CEACAM1 and CEACAM6 allowed bacterial internalization without triggering production of reactive oxygen species or release of toxic granule proteins [212]. However, all of the studies focusing on neutrophil responses to Opa-CEACAM binding have been done exclusively with the commonly studied N. gonorrhoeae strains MS11 [196,199,207,212,258] or FA1090 [209,210]. The presence of naturally occurring gonococcal phase variants that either do or do not express Opa proteins, obtained from a single low-passage clinical specimen, allowed us to establish whether the CEACAM binding specificity of primary LPCIs elicited cellular responses reminiscent of that seen with the recombinant strains. Isolate 4034B expresses Opa (Fig. 4-1, from hereon referred to as the Opa+ isolate) and shows binding to CEACAM1, CEACAM3, CEACAM5 and CEACAM6 (Fig 4-3), while 4034C lacks Opa expression (Fig. 4-1, from hereon referred to as the Opa- isolate), and does not bind CEACAM receptors (Fig. 4-3). When I assessed bacterial association of these two variants with human PMNs, the Opa+ isolate was bound and internalized to greater extent than was the Opa- isolate (Fig. 4-5A,B). Moreover, the Opa+ isolate was killed faster than was the Opa- strain (Fig. 4-5C). Next, we assessed activation of neutrophil antimicrobial responses after infection with these two phenotypic variants. The Opa+ variant activated production of higher levels of reactive oxygen species (ROS) than did the variant that lacks CEACAM binding capacity (Fig. 4-6A,B). The Opa+ variant also drives a robust degranulation of both primary and secondary granules, evident by the release of primary (elastase) and secondary (lactoferrin) granule contents, while infection with the Opa- variant did not lead to increased release of any granule proteins over uninfected controls (Fig. 4-6C).

100

101 Figure 4-4. Male and female isolates show no difference in CEACAM-binding profiles. (A) Whole cell dot blot of the original (mixed colony phenotype) low-passage clinical specimens. Gonococci were immobilized on nitrocellulose filters and allowed to bind soluble CEACAM1-Fc, followed by AP-conjugated goat-anti-human IgG and substrate development. - Control strains expressing no Opa (Opa ) and Opa57 are indicated on the top left. (B) Frequency of binding to indicated CEACAMs based on original low-passage clinical strains isolated from males or females, as determined by CEACAM1-Fc whole cell dot blot and 96-well binding assay. The binding frequency for combined isolates is shown above the bars.

102

Figure 4-5. CEACAM-binding LPCI is readily phagocytosed by human neutrophils. (A) Human neutrophils were infected with primary specimen-derived phase variants 4034B (Opa+) and 4034C (Opa-). Cells were fixed 30 min post-infection and visualized by staining for actin with Texas Red-phalloidin, and bacteria (UTR101) are shown in green. Note that bacteria within the image depicting the Opa- strain are rarely associated with the PMNs, whereas the Opa+ bacteria accumulate within the cells. Representative of 3 independent experiments. 63X Magnification. (B) Intracellular and total PMN-associated bacteria were differentially stained, and quantified via immunofluorescence microscopy. N=3, error bars represent SEM. One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, *P<0.05, **P<0.01. (C) Human PMNs kill Opa- and Opa+ isolates with different kinetics. Adherent PMNs were infected with either Opa- or Opa+ N. gonorrhoeae at an MOI=1. Bacterial survival over time was evaluated as CFUs present in PMN lysates at each time point relative to bacterial CFUs present at time 0. N=3, error bars represent SEM.

103 While CEACAMs are normally expressed on the surface of neutrophils, they also comprise a major component of neutrophil secondary granules. Consequently, the Opa- dependent neutrophil degranulation also increased expression of CEACAMs on the neutrophil surface (Fig. 4-6D), whereas infection with Opa- strain did not. I recently revealed that CEACAM3 engagement activates a pro-inflammatory transcriptional program in neutrophils, which results in production of pro-inflammatory cytokines via a signalling pathway that is independent of bacterial phagocytosis [258]. Consistent with this, infection with the Opa+ phase variant led to significantly higher levels of both TNF and IL-8 (Fig. 4-6E). Considered together, these results indicate that the CEACAM binding phenotype of primary LPCIs confers cell association and cellular response outcomes reflecting that seen with commonly used recombinant strains, and consistent with the expression of Opa variants that bind to CEACAM3 on neutrophils being detrimental to the gonococci.

104

105 Figure 4-6. Neutrophil bactericidal and inflammatory responses to phenotypic variants of N. gonorrhoeae. Human PMNs were infected with 4034B (Opa+) and 4034C (Opa-) at MOI 10. Neutrophil oxidative burst, degranulation, and cytokine release were measured as described in Materials and Methods. (A-B) Oxidative burst response to N. gonorrhoeae infection, illustrating the kinetics of response from one representative donor (A) and the mean with standard error (SEM) calculated based upon independent experiments with 3 different donors (B) One-Way ANOVA (with Tukey’s post-test) was performed for relevant samples, *P<0.05, **P<0.01. (C) Elastase and lactoferrin release in response to 4034B and 4034 C is shown a percentage (%) release, calculated as the amount of the protein in the supernatant divided by the amount released after PMA treatment. N=3, error bars represent SEM. (D) Flow cytometric analysis of CEACAM expression on human PMN cell surface. After infection with Opa+ or Opa- variant, the PMNs were fixed and stained with CEACAM-PE the MFI from one representative donor is shown. (E) Human neutrophils infected with Opa+ N. gonorrhoeae show increased levels of IL- 8 and TNF relative to PMNs infected with Opa- bacteria. Unpaired student t test was performed for relevant samples, **P<0.01, ***P<0.001.

106 4.5 DISCUSSION

N. gonorrhoeae is highly adapted to life in humans. Its narrow host range is largely defined by its adhesin proteins and immune evasion mechanisms that are specific for human cellular receptors and serum proteins [141]. Pilus facilitates initial bacterial attachment to the urogenital mucosa [17], where other adhesins can then confer a more tight secondary binding to epithelial-expressed receptors [7,81,141,293]. Of these, the Opa proteins mediate tight adherence and transcytosis to the subepithelial space [30,226]. The phase variability of opa and pili genes allows N. gonorrhoeae to change the patterns of expression of these adhesins randomly. Consequently, during a natural infection there may exist a mixed population of bacteria varying in piliated phenotype and/or the number and types of Opa variants expressed. This ongoing diversification of phenotypes is balanced by ongoing phenotypic selection of variants expressing adhesins or other factors that facilitate infection within an individual and/or within a particular tissue-specific niche. In vitro cell culture and primary cell-based systems clearly established that most Opa variants can bind one or more human CEACAM, and transgenic mouse-based studies corroborate the importance of this binding for the establishment of infection [200,201]. Experimental human urethral model studies also suggest the importance of Opa proteins and pilus [18-20,218-220,294], yet the binding specificity of Opa variants expressed during human infection has not been addressed until this study. The frequent phase-variable switching on and off of pilus and Opa expression necessitated that isolates be minimally passaged before phenotypic studies. To achieve this, we obtained primary LPCIs collected as part of a longitudinal study of commercial sex workers and STD clinic patients in Nairobi, Kenya. The analysis of pilus and Opa expression showed that the majority of the isolates expressed one or more Opa variants. All eleven chromosomally-encoded opa alleles are constitutively transcribed regardless of whether their respective translated proteins are phase-varied on or off [295]. Moreover, due to the hypervariable nature of the surface-exposed loops of each Opa variant, their binding specificity cannot be inferred by protein sequence. Since a bioinformatics-based approach was not feasible, and since the presence or absence of binding function is more important than the specific Opa alleles that confer binding, we sought to characterize the CEACAM binding phenotype of each isolate. To this end, Dr. Wong developed a high-throughput binding assay that allows quantitative assessment of CEACAM binding specificity. This revealed that most isolates bound both CEACAM1 and CEACAM5, perhaps reflecting the need for transmission between niches

107 expressing these two receptors. However, when considered more closely, it is noteworthy that all isolates from men bound CEACAM5 whereas only two-thirds of the isolates from women did so. Interesting in this regard, squamous cells of the lower genital tract tend to express CEACAM5 whereas columnar epithelial cells of the female endocervical and upper genital tract instead express CEACAM1 [36]. While CEACAM family expression within the male urethra has not been mapped, the difference in receptor specificity of recovered isolates makes it enticing to consider whether there is selection for CEACAM5 binding at this site. In contrast to the selection for CEACAM1 and CEACAM5 binding, we observed an apparent selection against binding to neutrophil CEACAM3 receptor, with only 27% of all isolates showing measurable binding to CEACAM3. Moreover, for those isolates that did bind to CEACAM3, the detected signal strength was generally weaker when compared to the other CEACAMs. This difference in binding cannot be explained by lower levels of CEACAM3 expression by the transfected cell line, as all CEACAMs were expressed on the surface at similar levels. Instead, the selection against CEACAM3 binding is consistent with binding to this receptor having detrimental consequences for the bacteria. Consistent with this, we observed that, in contrast to the Opa- variant, Opa+ phase variants that bound human neutrophils were more effectively engulfed and elicited a greater oxidative burst, degranulation and inflammatory cytokine-expression response. Curiously, several isolates were able to bind to one or more CEACAMs while seemingly lacking Opa expression. Whether this stems from their expression of Opa variants that are not recognized by the available Opa-specific monoclonal antibodies and/or from CEACAM binding occurring in an Opa-independent manner, as has recently been reported for N. meningitidis [296], will require further study of these isolates. Nevertheless, CEACAM binding defines the phenotype of these strains, regardless of any such antigenic differences. This study highlights the exquisite interplay of host-pathogen interactions that allows N. gonorrhoeae to persist in a human population. The ongoing phase variability of 11 Opa proteins allows their randomized expression, yet human infection selects for bacterial variants that specifically bind CEACAM1 and CEACAM5, but not CEACAM3. The selective pressure required to drive a preference between the human CEACAMs despite the high sequence and structural similarity of their respective bacterial binding N-domains [198,202] is a testament to the ongoing evolutionary dance between humans and our intimately associated neisserial partners.

108 5 General Discussion Neutrophils have long been considered to be a primary defense against infection due to their rapid response and ability to mobilize potent antimicrobial responses. However, they are also recognized as having the potential to provoke substantial tissue damage because many of their antimicrobial products are highly toxic to host cells. More recently, it has begun to be appreciated that PMNs may also help to regulate the emerging inflammatory response, contribute to adaptive responses, and play a role in resolution of inflammation [97,99]. Despite this, neutrophils’ molecular biology remains woefully understudied. This firstly stems from difficulties in the genetic manipulation of this cell type, the lack of good cellular models, and the short lifespan of primary cells when maintained ex vivo. In addition, it has been often assumed that molecular signaling pathways in a neutrophil would parallel those of a macrophage. However since these cells have significantly non-overlapping functions in the innate immune response, the comparisons should be drawn with caution. A variety of different bacterial and viral pathogens target CEACAM receptors to establish infection [77], highlighting their importance for microbial association with mucosal tissues. Indeed, the impressive evolutionary diversity of CEACAMs in different vertebrate species is believed to be driven by ongoing selection to avoid pathogen targeting. This is evident by the sequence variation among surface-exposed residues of the family’s evolutionarily progenitor, CEACAM1, but is more impressively highlighted by the frequent gene duplication events that leads to the diversity of CEACAM family members among different species (ranging from a single CEACAM1 in rabbits, to 29 CEACAM1-related genes in humans and 100’s of copies in bats) [62]. In each case, a single gene is presumed to retain the original cell-inhibitory CEACAM1 function conferred by the cytoplasmic ITIM motif, while the others become differentially expressed during development and/or gain alternate functions. For example, the GPI-anchors of CEACAM5 and CEACAM6 in humans have been proposed to allow ongoing shedding of these receptors from the surface of intestinal epithelial cells so as to prevent CEACAM1-mediated colonization of the mucosal surface [297]. In most species, one or more of the duplicated alleles contain an ITAM instead of an ITIM, implying an immune function similar to that described for CEACAM3 in humans [62]. N. gonorrhoeae has tended to be the paradigm used to understand how CEACAM binding affects the cellular response [29]. This stems, in part, from the fact that gonococci express Opa protein variants with the potential to bind various combinations of CEACAMs,

109 making the outcome of human cell binding dependent on both the repertoire of CEACAMs expressed and the Opa variant’s binding specificity. Moreover, CEACAM-dependent interactions seem to play a role in each stage of gonococcal infection and disease. While most interactions appear to benefit the bacteria, either by providing a convenient molecular handle by which to adhere to or penetrate across the mucosal epithelia [30,201,206] or to suppress the immune response [45,51,93], the association with CEACAM3 is difficult to see as advantageous for the pathogen. When considered in the context of the evolutionary diversification of CEACAMs in other species, it seems most reasonable that the selective pressure of well-adapted pathogens with substantial impact on human health, including life threatening childhood infections (H. influenzae and N. meningitidis) and the ability to cause female infertility (N. gonorrhoeae), has led to the development of a ‘decoy’ receptor with the ability to elicit a rapid innate immune response to combat infection. My studies have aimed to understand the protective and pathogenic consequences of this adaptation. 5.1 CEABAC Mice: The challenges in studying the biology of neutrophil-restricted CEACAM3 are closely linked to the challenges of studying neutrophils in general, as this short-lived cell type is resistant to genetic manipulation. A previous graduate student in our laboratory has established mouse promyelocytic cell lines (MPROs), which can be differentiated into neutrophil-like cells, expressing individual human CEACAM receptors. This approach allowed us to establish that human CEACAM function was maintained within mouse cells. However, expression of CEACAMs from constitutively active promoter leads to receptor over-expression, especially problematic in the case of CEACAM3, which is present at low levels in resting PMNs, and likely disrupted trafficking of CEACAMs to their proper PMN granules. Therefore, I chose to test the relevance of CEABAC mouse-derived neutrophils to investigate N. gonorrhoeae – neutrophil interactions. CEABAC mice express human CEACAM5 and CEACAM6 on epithelial cells, and CEACAM3 and CEACAM6 on neutrophils, each under their native promoters [234]. CEACAM3 and CEACAM6 expression and localization in PMNs from CEABAC mice reflect what we see in humans cells, and the responses of CEABAC neutrophils infected with Opa+ N. gonorrhoeae closely reflected what was seen in human cells. This data gave us confidence that CEABAC mice are good model for the proposed work.

110 5.2 CEACAM-dependent Transcriptional Response: Considering the emerging appreciation that neutrophils can alter their response depending upon the threat to which they are exposed, I sought to determine whether CEACAM expression impacted how neutrophils respond to N. gonorrhoeae. To do this, I compared transcriptional response of N. gonorrhoeae-infected WT and CEABAC PMNs to uninfected cells by full genome microarray. The general pattern of genes expressed in response to N. gonorrhoeae was similar in the WT and CEABAC animals, however two categories of transcriptional response were apparent. In the first group are genes that are induced to a similar level in WT and CEABAC PMNs. Of these, the largest functional classes of genes are those involved in the regulation of inflammation. In the second group are genes showing differential pattern of expression between CEABAC and WT PMNs. CEABAC neutrophils displayed a marked up-regulation of acute inflammatory cytokine expression. I inferred that the cumulative effect of these changes in gene expression would be a heightened pro-inflammatory cytokine response when the gonococcal Opa proteins engage the neutrophil-expressed CEACAM3. Consistent with this, significant increases in chemokine mRNA was evident by qPCR and corresponding protein levels were observed in supernatants from CEABAC PMNs infected with Opa+ bacteria, compared to supernatants from CEABAC PMNs infected with Opa- bacteria. Interestingly, I observed that phagocytosis and production of ROS are not essential for pro- inflammatory cytokine production. At the same time, I showed that bacterial engulfment on its own is not sufficient for cytokine response, as the inflammatory response could be inhibited without affecting the rates of bacterial phagocytosis. To understand the implications of these effects during in vivo infection, I measured the relative contribution of neutrophil CEACAM3 on inflammation using a subcutaneous air-pouch model, which has classically been used to measure inflammatory effects of drugs or other compounds. The air pouch experiments showed that the CEACAM-dependent association between N. gonorrhoeae and resident neutrophils promotes aggressive neutrophil recruitment, presumably due to the establishment of a chemotactic gradient. Consistent with this, the pro- inflammatory chemokines MIP-1α, MIP-2, KC, and IL-1β were all increased in Opa+ N. gonorrhoeae-infected air pouches in CEABAC mice relative to that seen in the infected WT littermates [251]. Together, my results established that engagement of CEACAM3 leads to establishment of a positive feedback loop that drives continuous neutrophil recruitment during N. gonorrhoeae

111 infection. This suggests that the successful CEACAM3-dependent recognition of Opa+ gonococci would prevent establishment of infection, either promoting rapid neutrophil-mediated clearance of the bacteria or driving the onset of urethritis. Although I show that CEACAM3 is involved in initial establishment of inflammatory environment, it is hard to imagine that the persistence of the inflammatory response can be attributed to CEACAM3 signalling alone. Neutrophil participation is critical to initialize timely resolution of inflammation via secretion of a number of lipid mediators, such as resolvins [97], so it would be interesting for future studies to ascertain whether or not these functions are disrupted during N. gonorrhoeae infection and whether or not this is beneficial to the host or the pathogen. 5.3 Specific Innate Immunity: Another interesting concept arising during my studies concerns the remarkable specificity of innate immunity. While this specificity is nowhere near the precision of the antigen receptors and adaptive immune response, it shows a much higher sophistication and selectivity of response than can be supposed considering the nature of main families of PRRs. It is the relatively recent discoveries of innate immune receptors with a much narrower range of ligands, such as C-type lectins [172] and now CEACAM3, that shed light on the discrimination exercised by the innate immune cells. Thus, only upon keeping in mind the complexity of multiple receptors and signalling networks acting together as a part of one system we can fully understand the sophistication of pathogen recognition. As with Dectin-1, CEACAM3 provides the ability to detect a class of microbes with the phagocytic and immune priming capacity of a phagocytic receptor. Consistent with the immune system’s need for two independent signals before unleashing a devastatingly potent response, CEACAM3 must be co-engaged with a PRR before the neutrophil will initiate its pro- inflammatory program. This relationship was not previously appreciated because the majority of work was done with Opa+ bacteria, which would provide both signals [258], or with antibody- coated latex particles that can phagocytosed without the second signal [195]. However, I observed that CEACAM-specific antibodies do not elicit a cytokine response in either human or CEABAC PMNs, yet concomitant treatment with LPS led to an IL-8 (human) or MIP-2 (mouse) response amplified to a level above that produced by either the CEACAM engagement or LPS alone.

112 5.4 CEACAM3-dependent Signalling: As previously mentioned, the pathology associated with symptomatic infection is caused by an overzealous immune response rather than direct bacterial-induced toxicity. While the initial inflammatory response presumably facilitates pathogen control, it might be beneficial to limit the amplitude and/or duration of inflammation so as to limit the unnecessary discomfort and tissue damage. Moreover, as discussed below, the vigorous inflammation might have important consequences on the development of adaptive and memory responses later on. For that purpose, I decided to investigate the signal transduction pathway downstream of CEACAM3 that leads to transcription activation, as well as phagocytic pathways downstream of CEACAM3, with aim of identifying mediators involved in the former but not the latter functions. My initial pharmacological- and biochemical-based assays showed that cytokine production was a result of NF-κB and p38 activation via a PKCδ and Tak1 serine/threonine kinase-dependent pathway triggered downstream of Syk tyrosine kinase. By interbreeding CEABAC mice with those lacking potential downstream effectors, I was able to dissect the relative contribution of the small Rho family GTPase Rac2 and the Bcl10-MALT1 complex in CEACAM3-mediated neutrophil responses. Interestingly, CEABAC PMNs that lack these effectors still readily engulf N. gonorrhoeae. However, Rac2 deficiency abrogated the CEACAM-dependent oxidative burst and seemed to be involved in both antimicrobial and inflammatory response. CEABAC PMNs deficient in either Bcl10 or MALT1 were still able to generate ROS and but were defective in their cytokine response. While I did not explore it in my studies, Bcl10 and MALT1 complex the with adaptor protein CARD9 in other myeloid cells, so I expect that the CEACAM3-dependent transcriptional response occurs via a CARD9-Bcl10- MALT1 complex. This, then, represents a signaling axis separate from one required for CEACAM3-dependent bacterial engulfment and killing. Since the role of the Bcl10-MALT1 complex remains controversial downstream of TLRs, I wondered whether these effectors might help facilitate the convergence of CEACAM3- and TLR-mediated signals. While there seemed to be a partial defect in TLR2 and TLR9 signalling in the Bcl10 and MALT1 knockouts, the LPS response was normal in both mouse lines suggesting that the TLR4 signals may occur without this pathway. Interesting in this regard, I observed that Bcl10 contributes to the synergistic response conferred when TLR4 and CEACAM3 are co-stimulated while MALT1 does not (Fig 5-1). Thus, based on these findings, it is tempting to suggest that a Bcl10 inhibitor could be used to ebb CEACAM3-mediated

113

Figure 5-1: Schematic representation of CEACAM3 signalling network. Opa-CEACAM3 interaction results in activation of Src PTK and phosphorylation of ITAM domain, and recruitment of Syk kinase. The signalling cascade that follows involves PKCδ, Bcl10-MALT1 complex, and Tak1 kinase and results in activation of p38 MAPK and NF-κB transcription factors, and cytokine secretion. In addition, Rac2 is also involved in cytokine signaling, however further experiments are needed to pinpoint its exact role in the cascade. I also propose that CEACAM3 is acting in synergy with TLR4 to fine tune the transcriptional response, hypothetically via a novel mechanism involving Bcl10. (Light gray indicates hypothetical interactions)

114 inflammation and associated immunopathology, while having minimal effect on PMNs antimicrobial functions, or cells ability to signal via other PRRs. 5.5 CEACAM Interactions: An interesting and important aspect of CEACAM3 signalling that was not considered in this thesis work is the potential effect of CEACAM1 co-engagement on CEACAM3 signalling. While CEABAC PMNs do express mouse CEACAM1, this receptor is not recognized by the neisserial Opa proteins, suggesting that it would not be co-engaged as would be expected in human neutrophils. When human CEACAM1 and CEACAM3 were co-expressed in the mouse- derived MPRO neutrophil cell line, CEACAM1 seemed to enhance CEACAM3-dependent signalling rather than functioning as a co-inhibitory receptor, as it does in other cell types [212]. However, the mechanism for this co-operation has not been elucidated. Additionally, as mentioned above, the cell line might not be the ideal model to study CEACAM1-CEACAM3 functional interaction, and the collaboration seen in cell culture needs to be confirmed in primary bone marrow derived PMNs for mice expressing human CEACAM1 as well as CEACAM3. Regardless, the effect of CEACAM1 in the context of neutrophils, both as a cell adhesion molecule and in the context of CEACAM3 signalling, does merit further investigation. 5.6 CEACAMs in Gonococcal Infection: While our current understanding of the contribution of CEACAMs to gonococcal infection stems from a combination of in vitro and, more recently, humanized transgenic mouse- based studies, we sought some indication of whether or not our emerging models reflect what occurs in humans. While classical study of the opacity phenotypes of gonococcal colonies isolated from patients with gonorrhea [219] and studies with male human volunteers receiving urethral inoculation with transparent colonies of N. gonorrhoeae [218,220] both suggest that Opa protein expression is important for the establishment and progression of infection, these studies were performed before the receptor specificity of Opa proteins was known. Given that each gonococcal strain has the potential to express Opa variants that bind to various combinations of CEACAM1, CEACAM3, CEACAM5 and/or CEACAM6, or to turn off Opa expression completely, our finding that 89% of all clinical isolates showed strong binding to epithelial CEACAM1 and/or CEACAM5 is consistent with these interactions providing a selective advantage for the gonococci during natural infection. In stark contrast, only 27% of isolates bound to CEACAM3, suggesting that there is selection against expression of Opa variants that bind this receptor. In order to confirm that the differential expression of Opa

115 variants in these clinical isolates conferred a phenotype similar to that seen when varying Opa protein expression in our recombinant laboratory strains, I compared human neutrophil responses to two clinical phase variants differing in their ability to bind CEACAMs. Consistent with our expectation, the isolate able to bind to CEACAMs including CEACAM3 was actively phagocytosed by the neutrophils, stimulated the production of reactive oxygen species and the release of granule proteins, and activated secretion of pro-inflammatory cytokines, whereas the other variant did not. When considered together, this study provides the first description of the CEACAM binding specificity expressed during naturally acquired infection, and supports a model wherein every gonococcal strain has the potential to express Opa proteins of any specificity, but the differential selection of Opa variants mediating attachment to different cell types in vivo is apparent. The natural (albeit ethically, technically and financially difficult) extension of these studies would be the infection of human volunteers with N. gonorrhoeae constitutively expressing Opa variants of known specificity to assess the effect on infection and immune pathogenesis. 5.7 Neutrophils and the Adaptive Response: One of the main hurdles in our battle with N. gonorrhoeae is the lack of protective immune response to infection. The antibody responses elicited by gonorrhea are weak and fade quickly [13,14], allowing individuals to become re-infected by the same strains of N. gonorrhoeae [12]. While high levels of antigenic and phase variation might hinder the development of effective antibody response, this idea does not explain the complete lack of immunological memory, particularly upon reinfection with strains expressing conserved surface antigens [12]. Although this phenomenon remains largely an enigma, recent studies attribute it to N. gonorrhoeae skewing the adaptive immune response from more long term protective

Th1/Th2 to inflammatory Th17 features [298]. A Th17 response is essential for an effective defense against a number of mucosal pathogens, and is characterized by enhanced mobilization and recruitment of neutrophils, increased epithelial barrier function, and epithelilal antimicrobial peptide secretion [299] However, during N. gonorrhoeae infection in mice, the gonococci can persist despite this response while benefiting (both during this and subsequent infections) from the lack of more effective adaptive response [300,301]. Although these elegant studies raise more questions then they answer, they are the first to suggest a mechanistic explanation for the dearth of gonococcal infection-induced immunity. However, these studies were performed in animals that lack human factors that play a role in N. gonorrhoeae infection, including

116 CEACAM3. It will be interesting to determine whether CEACAM3 binding influences, either through exacerbation or alleviation, this immune regulatory effect. In the context of immunity, it is important to consider that neutrophils are widely recognized as a multifaceted cell type that can act as an effector in both innate and adaptive immunoregulatory networks [98,302]. Despite the obvious participation of neutrophils in gonorrhea, this aspect of neutrophil biology has been virtually ignored in gonococcal research. Considering the evidence presented in this thesis, it seems critical to re-evaluate the neutrophils’ role in N. gonorrhoeae-induced immune responses. For instance, under certain circumstances neutrophils have been shown to take on APC-like characteristics, function like DCs, or differentiate into macrophages [302]. Most of these studies are based on in vitro work, and the biological function of neutrophil plasticity and molecular mechanisms involved are yet to be determined, however the fact that they can occur is provocative. Moreover, neutrophils can establish cross talk with cells of both innate and adaptive immune response, and can be involved in enhancement of macrophage function [302], recruitment of Th17 T cells [120], and modulation B cell function [120,133,302]. Considering the dominance of neutrophil response in N. gonorrhoeae infection, some or all of these could play an important role in the resulting immune response. Since CEACAM3 is able to activate a robust transcriptional response in PMNs, I hypothesize that it would be heavily involved in other aspects of immune response modulation. The recent development of models of N. gonorrhoeae asymptomatic lower genital tract colonization and ascending PID in ‘humanized’ mice provides exciting potential to further our understanding neutrophil biology in the context of gonococcal infection. 5.8 Conclusions: It is now clear that CEACAM3 functions as a decoy receptor that allows the capture and killing of CEACAM-targeting microbes. The apparent involvement of CEACAM3 in pro- inflammatory signaling establishes a role for neutrophils beyond basic microbial killing, and places CEACAM3 as both a potential contributor to an accelerated response to N. gonorrhoeae infection and, subsequently, to the immunopathology associated with the gonococcal disease. My studies have aimed to define the molecular mechanisms involved in these CEACAM3- mediated responses, with the hope that this will give us the ability to manipulate the pathological inflammatory response seen in N. gonorrhoeae infection, and contribute insight as to how the innate immune response can adapt to highly adapted microbial pathogens. The evolutionary advent of CEACAM3 thus reflects the latest step in the ongoing dance between

117 Neisseria and their only natural host, providing a snare that mobilizes our most potently bactericidal cells against this stealthy invader, and directs the course of the immune response.

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