lactobacillus rhamnosus gr-1 supernatant on endotoxin ... · ii the effect of lactobacillus...

The Effect of Lactobacillus rhamnosus GR-1 Supernatant on Endotoxin-Induced Cytokine Secretion

in Human Myometrial Cells

by

Bona Kim

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology

University of Toronto

© Copyright Bona Kim 2017

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The Effect of Lactobacillus rhamnosus GR-1 Supernatant on Endotoxin-Induced Cytokine Secretion in Human Myometrial Cells

Bona Kim Master of Science

Department of Physiology

University of Toronto

2017 ABSTRACT

Prophylactic administration of probiotic Lactobacillus rhamnosus GR-1 supernatant

(GR1SN) was shown to prevent lipopolysaccharide (LPS)-induced preterm birth (PTB) in mice

by suppressing pro-inflammatory cytokine expression in uterine tissues in vivo. The aim of this

thesis is to elucidate the effects of GR1SN in human myometrial cells in vitro, where I

hypothesize it will suppress LPS-induced cytokine secretion. Here I demonstrate that PBS-based

GR1SN contains protein moieties that induce endotoxin tolerance-like phenotypes in myometrial

cells to suppress secretion of pro-inflammatory factors following LPS stimulus. The observed

effects are unique to a GR1SN-myometrial cell interaction, and cannot be replicated using

supernatant derived from non-GR-1 lactobacilli species. However, in vivo, GR1SN-PBS does not

delay the onset of LPS-induced PTB in pregnant CD-1 mice under the experimental conditions

used. Through this project, I have highlighted the unique capacity of protein moieties secreted by

L. rhamnosus GR-1 to inhibit inflammatory signals in human myometrial cells.

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ACKNOWLEDGEMENTS Writing this thesis has not only provided me the opportunity to summarize the work I did through my master’s project but also reflect on the people that have motivated me through it all. I would like to firstly express my sincerest gratitude to my supervisor, Dr. Stephen Lye, for trusting me to conduct quality research and for providing me with the optimal environment in which to do so. To Dr. Oksana Shynlova, for your unfaltering guidance and academic support, for respecting all my tangential speculations and bringing them together to a tangible experimental path. To my advisory committee members: Dr. Alan Bocking, Dr. Kevin Kain, and Dr. Scott Gray-Owen. Your individual expertise, perspectives, and suggestions are what shaped my project. Thank you for your thought-provoking ideas and discussions. To all the members of the Lye lab: Dr. Caroline Dunk, Dr. Jianhong Zhang, Dr. Mark Kibschull, Anna Dorogin, Ela Matysiak-Zablocki, who were always available to help with any question I had. A special thanks to Dr. Lubna Nadeem, who one day mentioned that “data will always be true – our role is simply to make sense of it”. Your words have provided me footing to stand on during times of scientific frustration, and will always continue to do so. To Tali Farine, Melissa Kwan, Tanya Tyagi, Justin Rondeau, and Jovian Wat – my sanity would not have survived the stress of failed experiments or inexplicable data without all of you. Thank you for reminding me that happiness and life continue to exist outside the walls of the lab, and for your input through the struggles of meticulously drafting emails. A warm thanks to Bev Bessey, for your assistance in scheduling meetings and for being on top of every deadline and reminder. My committee meetings and defense would not have happened without your help. Finally, an endless thanks to my mum. Thank you for all your patience, your understanding, and your support in the choices I have made during my master’s project. As I conclude this chapter, I would like to once again thank everyone who has made my journey enjoyable. It has been an incredible two years, during which I matured tremendously both academically and personally. Each of you have had a role in that process, and I would like to let you all know once again that it was greatly appreciated. Thank you.

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TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

LIST OF TABLES vii

LIST OF FIGURES viii

LIST OF ABBREVIATIONS x

CHAPTER 1: LITERATURE REVIEW

1.1 Preterm Labour: An Overview 1

1.2 Inflammation and Labour 2

1.3 Toll-Like Receptors 3 1.3.1 Toll-Like Receptors and Labour 4

1.4 Cytokines, Chemokines and Labour 5 1.4.1 Pro-inflammatory Cytokines

1.4.1.1 Tumour Necrosis Factor Alpha 6 1.4.1.2 Interleukin-1 beta 6 1.4.1.3 Interleukin-6 7

1.4.2 Anti-inflammatory Cytokines 8 1.4.2.1 Interleukin-4 8

1.4.2.2 Interleukin-10 9 1.4.2.3 Interleukin-13 9

1.4.3 Colony-Stimulating Factor 10

1.4.4 Chemokines 1.4.4.1 Interleukin-8 11 1.4.4.2 Monocyte Chemoattractant Protein-1 11

1.4.5 Other Mediators of Labour 12

1.5 Characterization of Vaginal Microbiota in Non-Pregnant Women 13

1.6 Characterization of Vaginal Microbiota in Pregnant Women 14

1.7 Probiotics 15 1.7.1 Beneficial effects of probiotic administration 16

1.8 Rationale & Hypothesis 1.8.1 Rationale 19 1.8.2 Hypothesis & Objectives 19

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CHAPTER 2: MATERIALS AND METHODS

A. IN VITRO EXPERIMENTS

2.1 MRS agar and broth preparation 20

2.2 Lactobacilli supernatant (SN) preparation and modification 2.2.1 MRS-based SN (GR1SN-MRS) 20 2.2.2 PBS-based SN (GR1SN-PBS) 20 2.2.3 GR1SN Size Fractionation 20

2.3 Macromolecular Detection and Fractionation in –SN 2.3.1 Protein Detection 21 2.3.2 Nucleic Acid Detection 21 2.3.3 Specific Elimination of GR1SN Macromolecule Function 22

2.4 In vitro cell culture 2.4.1 Primary human myometrial tissue collection 22 2.4.2 hTERT-HM culture 23 2.4.3 TLR agonist kit 23

2.5 Cytokine Quantification Assays 2.5.1 Enzyme Linked Immunosorbent Assay 24 2.5.2 Human 10-multiplex Cytokine Assay (Bio-Plex) 24

B. IN VIVO EXPERIMENTS

2.6 Animals 25

2.7 LPS Preparation 25

2.8 Intraperitoneal Injections 25

2.9 Animal Groups 25

C. STATISTICAL ANALYSIS 26

CHAPTER 3: RESULTS


3.1 Establishing an appropriate cell line & inflammatory stimulus 27

3.2 GR1SN-MRS pre-treatment attenuates LPS-induced inflammatory responses in hTERT-HM cells 27

3.2.1 Identification of macromolecular compounds in GR1SN-MRS 28

3.3 MRS broth influences myometrial inflammation 32

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3.4 GR1SN-PBS pre-treatment can suppress LPS-induced cytokine and chemokine secretions by hTERT-HM cells 35

3.4.1 Identification of functionally active macromolecules in GR1SN-PBS 35 3.4.2 Size Fractionation of GR1SN-PBS 38

3.5 Low-dose TLR agonist pre-treatment attenuates LPS-induced inflammatory responses in hTERT-HM cells 42

3.5.1 Low-dose LPS and Pam3CSK pre-treatment can suppress LPS-induced cytokine secretion 44

3.6 Cytokine regulation in myometrial cells by GR1SN was unique as compared to four non-L. rhamnosus GR-1 lactobacilli species 49


3.7 In vivo effects of GR1SN-MRS pre-treatment to delay the onset of LPS-induced PTL 51

3.8 In vivo effects of GR1SN-PBS pre-treatment to delay the onset of LPS-induced PTL 55

CHAPTER 4: DISCUSSION

4.1 In vitro effects of GR1SN pre-treatment on cytokine suppression in hTERT-HM cells stimulated with LPS 54

4.1.1 Endotoxin Tolerance in hTERT-HM cells 54 4.1.2 Effect of MRS broth on inflammatory signals in hTERT-HM cells 60

4.2 In vivo functional discrepancies of GR1SN effect 62

4.2.1 Intrauterine vs. intraperitoneal mode of LPS infusion 63 4.2.2 Tissue specificity of Lactobacilli effect 65

4.2.3 Compositional differences between GR1SN-MRS and GR1SN-PBS 66

4.3 Future Directions 67

REFERENCES 69

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LIST OF TABLES

Table 1. 10-plex BioPlex cytokine assay of media conditioned by hTERT-HM cells treated with GR1SN-MRS 43

Table 2. The effect of prophylactic GR1SN-PBS on delivery rates of LPS-induced preterm birth in pregnant CD-1 mice. 52

Table 3. The effect of prophylactic GR1SN-PBS on delivery rates of LPS-induced preterm birth in pregnant CD-1 mice. 53

Table 4. Summary of current and previous in vivo results showing rates of LPS-induced PTB following prophylactic GR1SN treatment. 63

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LIST OF FIGURES

Figure 1. Cytokine secretion by primary human myometrial cells (#1-5) in response to LPS

stimulus 29

Figure 2. Comparative dose and time responses following LPS administration in hTERT-HM

cells 30

Figure 3. Effect of GR1SN-MRS treatment on hTERT-HM cell cytokine secretion following

LPS stimulus 31

Figure 4. Characterizing macromolecules in GR1SN-MRS 33

Figure 5. Relative cytokine secretions by hTERT-HM cells pre-treated with modified GR1SN-

MRS prior to LPS stimulus 34

Figure 6. Effect of MRS broth on cytokine secretion in hTERT-HM 36

Figure 7. Effect of GR1SN-PBS treatment on cytokine suppression following LPS stimulus 37

Figure 8. Quantifying proteins in GR1SN-PBS 39

Figure 9. Effect of modified GR1SN-PBS on LPS-induced cytokine secretions 40

Figure 10. Effect of hTERT-HM pre-treatment with various GR1SN-PBS size fractionation on

LPS-induced cytokine secretions 41

Figure 11. Effect of low-dose LPS pre-treatment prior to high-dose LPS stimulus on cytokine

secretions by hTERT-HM cells 45

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Figure 12. Effect of TLR1/2 agonist (Pam3CSK) dose on cytokine secretion by hTERT-HM

cells 46

Figure 13. Effect of TLR2/6 agonist (FSL-1) dose on cytokine secretion by hTERT-HM cells 47

Figure 14. Effect of low-dose Pam3CSK pre-treatment prior to high-dose LPS stimulus on

cytokine secretions by hTERT-HM cells 48

Figure 15. Effect of five different lactobacilli strains on cytokine suppression in hTERT-HM

cells stimulated with LPS 50

x

LIST OF ABBREVIATIONS ANOVA Analysis of Variance

AP-1 Activator Protein 1

BCA Bicinchoninic Acid

BN Benzonase Nuclease

BV Bacterial Vaginosis

CCL Chemokine (C-C motif) ligand

CM Conditioned Media

COX Cyclooxygenase

CST Community State Type

CXCL Chemokine (CXC motif) Ligand

DMEM/F-12 Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12

DNA Deoxyribonucleic acid

EDTA Ethylenediaminete traacetic acid

ELISA Enzyme Linked Immunosorbent Assay

G-CSF Granulocyte Colony Stimulating Factor

GD Gestational Day

GM-CSF Granulocyte Macrophage Colony-Stimulating Factor

GPCR G protein-coupled receptor

GPI Glycophosphatidylinositol

GR1SN L. rhamnosus GR-1 supernatant

GR1SN-MRS MRS-based GR1SN

GR1SN-PBS PBS-based GR1SN

hBD-3 Human b-Defensin-3

hTERT-HM Human Myometrial Smooth Muscle Cell Line Immortalized with Human

Telomerase Reverse Transcriptase

IFN Interferon

IL Interleukin

ITS Insulin-Transferrin-Selenium

IUGR Intrauterine Growth Restriction

JAK/STAT Janus Kinase and Signal Transducers and Activators of Transcription

xi

kDa Kilodalton

L. rhamnosus Lactobacillus rhamnosus

LPS Lipopolysaccharide

LTA Lipotoichteic acid

M-CSF Macrophage Colony Stimulating Factor

MAMP Microbe-Associated Molecular Pattern

MAPK Mitogen-Activated Protein Kinase

MCP-1 Monocyte Chemoattractant Protein-1

MD2 Lymphocyte Antigen 96

MLC Myosin Light Chain

MMP Matrix Metalloproteinase

mRNA Messenger RNA

MRS de Man, Rogosa and Sharpe

NF-kB Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells

NK Natural Killer

OXTR Oxytocin Receptor

PAMP Pathogen-Associated Molecular Pattern

PBS Phosphate Buffered Saline

PG Prostaglandin

PGE2 Prostaglandin E2

PGF2a Prostaglandin F2a

pH Power of Hydrogen

PI3K Phosphoinositide 3-kinase

pPROM Preterm Premature Rupture of Membranes

PRR Pathogen Recognition Receptor

PS80 Polysorbate 80

PTB Preterm Birth

PTL Preterm Labour

RNA Ribonucleic Acid

SEM Standard Error Mean

SFM Serum Free Media

xii

SOCS Suppressor of Cytokine Signalling

STAT Signal Transducers and Activators of Transcription

TAE Tris-Buffer, Acetic Acid, EDTA

TLR Toll-Like Receptor

TNIL Term Not-in-Labour

TNF Tumour Necrosis Factor

TOLLIP Toll Interacting Protein

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CHAPTER 1: LITERATURE REVIEW

1.1 Preterm labour: an overview

Preterm delivery, birth of an infant prior to 37 weeks gestation, is a global medical

problem that affects millions of mothers and infants annually. Thirty percent of these cases are

medically induced to alleviate predisposing gestational complications such as preeclampsia or

intrauterine growth restriction (IUGR) (Agrawal and Hirsch, 2012), but other cases of preterm

labor (PTL) are idiopathic and occur spontaneously. One known cause of spontaneous PTL

(sPTL) is intrauterine infection, found in 40% of all preterm births (PTB) (Agrawal and Hirsch,

2012). Additionally, correlations have been observed between PTB and maternal stress,

shortened cervix, uterine insufficiencies, uterine over-distension, and preterm premature rupture

of fetal membranes (pPROM), (Romero et al., 2014a) making the treatment of PTB challenging

to target.

Current therapeutic interventions for PTB include prostaglandin (PG) synthesis inhibitors

(indomethacin) and calcium channel blockers (nifedipine) as more common tocyolytics, as well

as oxytocin receptor (OXTR) antagonists (atoxiban), magnesium sulfate, or nitric oxide donors

as less commonly used options. However, these drugs are all associated with high maternal and

fetal side effects, and do not target the underlying cause that initiates the premature contractions

(Simhan and Caritis, 2017). The use of antibiotics as adjunct therapy for women with urogenital

tract infections and intact membranes has been reviewed in a 2013 meta-analysis, indicating that

though antibiotics result in reduced infection, they are ineffective at preventing or delaying PTL

(Flenady et al., 2013). Interestingly, recent studies have indicated an association between vaginal

dysbiosis and infectious PTL, suggesting a protective role of the vaginal microbiome when in

homeostasis, offering a potential target for future clinical interventions. Today, the main goal of

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tocolytic treatment is to delay labour sufficiently to allow the administration of antenatal drugs

like corticosteroids to improve neonatal outcome. As such, current medical advances have

focused on the survival of the preterm neonate, which are costly and require technologies that are

limited to urban centres, leaving a large proportion of those affected by PTB in developing

countries unable to gain access to proper treatment. To achieve global equality in healthcare and

improve current management of PTB, novel therapeutic methods to address PTL are urgently

required.

1.2 Inflammation and labour

Over the past decade, research has demonstrated that maternal inflammation is a key

regulatory event that precipitates both pathological and physiological labour. In term labour,

stimulants such as functional progesterone withdrawal, uterine stretch, and fetal and maternal

endocrine stimulation activate local inflammatory processes which involve production of

cytokines and chemokines which function in autocrine, paracrine, or endocrine mechanisms to

recruit peripheral leukocytes, (Shynlova et al., 2012, 2013a, b) and activate local transcription

factors such as NF-kB and AP-1. These pathways increase production of labour-associated

molecules like matrix metalloproteinases (MMP), prostaglandins (PG), oxytocin, and gap

junction proteins like connexin-43 (Cx-43) that induce cervical softening and dilation, fetal

membrane rupture, and synchronized myometrial contractions to expel the fetus from the uterus.

In cases of pathological PTL, these same pathways can be activated upon host recognition of

pathogens Escherichia coli, Ureaplasma urealyticum, Mycoplasma hominis, and Streptococcus

agalactiae (Peltier et al., 2010). The inflammatory pathways that orchestrate labour are

comprised of more than 50 chemokines that instigate local migration of maternal peripheral

blood leukocytes into uterine tissue (Mackay, 2001). Leukocytes include five subtypes; namely

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neutrophils, eosinophils, basophils, monocytes and lymphocytes. Following activation,

neutrophils become degranulated and trigger angiogenesis or angiostasis (Salamonsen et al.,

2002), and monocytes differentiate into macrophages, which phagocytose apoptotic cellular

debris or pathogens. All leukocytes promote further production of chemokines and attract more

leukocytes to amplify the inflammatory response, explaining the difficulty in reversing labour

after its initiation. Although the complete spectrum of stimulants that activate maternal

inflammation prior to labour has yet to be elucidated, recognition of pathogens within the genital

tract by host pathogen recognition receptors (PRRs) like toll-like receptors (TLRs) play an

important role in inducing infectious PTL.

1.3 Toll-like Receptors

TLRs are a transmembrane family of PRR proteins that recognize various endogenous

and microbial ligands (i.e. microbial-associated molecular patterns – MAMP) to activate immune

responses in host cells. Often, they function as homo- or hetero-dimers to activate downstream

pathways and transcription factors such as NF-kB, which regulate expression of inflammatory

cytokines and chemokines. In humans, 10 TLRs have been detected. TLRs 1, 2, and 6 function

as membrane bound TLR1/2 or TLR2/6 heterodimers to recognize tri- or di-acylated lipoproteins

of bacteria. TLR3 is localized intracellularly to recognize viral double stranded (ds)RNA such as

PolyI:C. TLR4 is a potent receptor for lipopolysaccharide (LPS), a component of gram-negative

bacteria wall, while TLR5 recognizes bacterial protein flagellin. TLR7 and TLR8 serve as

receptors for single stranded (ss)RNA, and TLR9 recognizes unmethylated CpG DNA

intracellularly. TLR10 agonists remain unknown to date.

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1.3.1 Toll-like receptors and labour

TLRs are associated with term and preterm labour, intrauterine infections, and pregnancy

complications through activation of immune responses in gestational tissue. In the human cervix,

an increased expression of TLR2 and TLR4 with decreased expression of TLR3 and TLR5 are

thought to be responsible in part for cervical remodelling at labour (Ekman-Ordeberg and

Dubicke, 2012). Decidual cells also express TLR1, TLR4, and TLR6 contributing to infection-

associated PTB (Patni et al., 2007). A recent study suggested alterations in tissue sensitivity

induced by epigenetic modifications that are associated with increased TLR2 and TLR9

expression in decidual cells isolated from women with spontaneous PTL (sPTL) (Walsh et al.,

2017). In the placenta, TLR2 and TLR4 have been detected on trophoblast cells, while

choriocarcinoma cells respond to exogenous ligands for TLR2, 3, 4 and 9 (Patni et al., 2007).

Interestingly, in placentae of preterm deliveries, the functional activity of TLRs characterized by

cytokine production was decreased, although TLR gene expression remained unaltered as

compared to term controls (Patni et al., 2015), indicating various alterations in both expression

and function of TLRs in relevance to labour. Amniotic epithelial cells also express TLR2 and

TLR4, which is significantly increased in term labouring samples as compared to term not in

labour. In the human myometrium, TLR2 and TLR4 are increased during term labour compared

to preterm, and TLR2 is highly expressed during labour than non-labouring states. (Youssef et

al., 2009)

The importance of TLR4 in regulation of parturition has been demonstrated in animal

studies, where TLR4 KO mice deliver on average 13 hours later than wild type controls, and

newborn pups show increased perinatal mortality. These mice demonstrate decreased

expressions of pro-inflammatory cytokines, namely interleukin (IL)-1β, IL-6, IL-12β, and tumor

necrosis factor (TNF)-α in placental and fetal membrane tissues when compared to control mice.

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(Wahid et al., 2015) Moreover, a TLR4 polymorphism (Asp299Gly) that is associated with

impaired TLR4 function in humans has been detected in higher prevalence in preterm infants

when compared to infants born at term. (Lorenz et al., 2002) These studies help outline the

contribution of TLRs, especially TLR4 to labour, and identified them as potential targets in

therapeutic intervention for prevention or treatment of PTB.

1.4 Cytokines, Chemokines and Labour

The activation of TLRs stimulates multiple downstream pathways that result in

expression of inflammatory cytokines, which are small extracellular signaling proteins including

interleukins (IL), interferons (IFN), and growth factors. Secreted by host cells, cytokines

function in auto-, para- or endocrine fashion by binding to target receptors. Pro-inflammatory

cytokines moderate the classical phenotypes of inflammation such as vascular dilation, increased

endothelial permeability, and leukocyte activation that regulate cellular apoptosis or pathogen

removal, while anti-inflammatory cytokines counteract these events to protect the inflamed

tissue. Although moderate levels of inflammation alleviate infection and promote pathogen

removal, prolonged inflammation can lead to excessive cellular apoptosis and cause tissue

damage. Near term, pro-inflammatory cytokines such as TNF-a, IL-1β, IL-6, and IL-8 (Winkler

et al., 2001; Kemp et al., 2002) and labour-mediating molecules like MMPs or PGs augment

cervical dilation, membrane rupture and myometrial contraction. Cytokine concentrations vary in

a tissue specific manner; the myometrium expresses increased concentrations of TNF-a, IL-1b,

and IL-6 during term labour and LPS-induced PTL, while PTL induced by progesterone

antagonist mifepristone demonstrated increased IL-1b and IL-6 expression. (Shynlova et al.,

2013a) The choriodecidua shows increased levels of CCL2, 4, 5, 8, and 10 in women prior to

PTL, (Hamilton et al., 2013) and increased expression of IL-8, IL-1b, IL-6, and TNF-a is

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detected in the cervix (Patni et al., 2007) and amniotic fluid. (Holst et al., 2011) Interestingly,

pro-inflammatory cytokines such as IL-1b, IL-6, and IL-8 are also increased in the maternal

plasma, (Torbé et al., 2007) indicating a systemic response to local signals.

1.4.1 Pro-inflammatory cytokines 1.4.1.1 Tumour necrosis factor alpha (TNF-a)

TNF-a is a major acute phase inflammatory cytokine, that is expressed by most cells.

Immune cells such as lymphocytes, macrophages, NK cells, neutrophils, mast cells, and

eosinophils produce both secreted and membrane-bound forms of TNF-a, which allow

intercellular communication to induce inflammation, cellular apoptosis, or proliferation.

(Christiaens et al., 2008; Kawano et al., 2012) During pregnancy, tight regulation of TNF-a

expression is required as unprecedented elevations of TNF-a have been associated with

increased risk of fetal loss, gestational diabetes, hypertensive syndromes such as preeclampsia,

and IUGR. (Brogin Moreli et al., 2012) During healthy pregnancies, low concentrations of TNF-

a are maintained in gestational tissue until a rapid increase during labour, (Hayashi et al., 2008;

Shynlova et al., 2013a) which stimulates prostaglandin and MMPs production in the cervix to

induce cervical ripening and in placental and myometrial tissues to induce membrane rupture and

myometrial contraction. (Christiaens et al., 2008)

1.4.1.2 Interleukin 1-beta (IL-1β)

IL-1β is an upstream regulator of inflammation that stimulates multiple downstream

pathways. In pregnancy, increased IL-1β expression has been detected in cervico-vaginal tissue

approaching labour (Heng et al., 2014) and shown to induce production of MMP, promoting

cervical ripening and dilation, while decreasing production of TIMP (endogenous inhibitors of

MMPs). (Patni et al., 2007) COX-2, a rate-limiting enzyme involved in prostaglandin synthesis,

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is also further induced by IL-1β. PGE2, in association with cytokines TNF-a and IL-1β are

powerful promoters of uterine contraction in the myometrium. High IL-1β production is also

detected in fetal amniochorion and decidua in both term and preterm labour regulating

membrane rupture. In the myometrium, IL-1β is also responsible for increased oxytocin secretion,

leading to increased concentrations of intracellular calcium. The consequence of these events are

powerful synchronized myometrial contractions, which expel the fetus.

Experiments in pregnant rhesus monkeys demonstrated that intra-amniotic infusions of

TNF-a and IL-1b can induce PTB within 1.1 days from point of injection, compared to 27.6

days in control monkeys (Sadowsky et al., 2006). It was observed that the administration of

TNF-a and IL-1b induced production of IL-8, IL-6, PGs, and MMP-9 in amniotic fluid, resulting

in an amplified inflammatory response and leukocyte infiltration (Sadowsky et al., 2006). In

contrast, treatment with IL-6 or IL-8 did not induce preterm myometrial contractions (treatment

to delivery interval was 21.9 days) or increased TNF-a and IL-1b expression in pregnant rhesus

monkeys, demonstrating an upstream regulation of IL-6 or IL-8 by TNF-a and IL-1b infusion

Knock-out experiments have further confirmed these findings as pregnant mice with TNF-a, IL-

1b double receptor knockouts were protected against local bacteria-induced PTL with reduced

myometrial production of PG synthesizing enzyme COX-2 (Hirsch et al., 2006), while IL-6 -/-

mice did not show resistance to bacteria-induced PTL. (Yoshimura and Hirsch, 2003)

1.4.1.3 Interleukin-6 (IL-6)

IL-6 is an inflammatory cytokine secreted by immune cells to induce an acute phase

inflammatory response along with TNF-a, and IL-1β. IL-6 stimulates differentiation and

maturation of immune cells such as B- and T-cells, as well as hematopoiesis in the bone marrow

(Robertson et al., 2010). In gestation, IL-6 is a critical element for the timely onset of parturition,

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regulating genes involved in PG-mediated uterine activation, (Papatheodorou et al., 2013) as

indicated by delayed labour in term IL-6 knockout mice as compared to wild type mice

(Robertson et al., 2010). No associations between IL-6 expression and gestational age have been

found; although increased IL-6 mRNA and protein expression has been characterized in the

laboring myometrium. (Elliott et al., 2000; Sharp et al., 2016) Specifically, IL-6 expression,

along with IL-1β and IL-8 is significantly higher in the lower uterine segment during term labour

as compared to PTL, but not in the upper segment. (Tattersall et al., 2008) IL-6 expression was

increased in fetal membrane, cervical, and decidual tissue during term and preterm labour with

and without infection, indicating its involvement independent of cause of labour onset. (Patni et

al., 2007) IL-6 induces OTXR expression on myometrial cell surface (Rauk et al., 2001)

although it has no direct effect on myometrial contraction or PG synthesis (Peltier, 2003).

1.4.2 Anti-inflammatory Cytokines

Anti-inflammatory cytokines function to support healthy pregnancies by playing a key

role in maintaining inflammatory homeostasis. These include cytokines such as IL-4, IL-10, and

IL-13, which are produced by immune cells and certain tissue types to respond to the effects of

pro-inflammatory cytokines (Wilczyński, 2005; Romero et al., 2006).


IL-4 is an anti-inflammatory cytokine that antagonizes production of pro-inflammatory

cytokines TNF-a, IL-1β and PGE2 by human macrophages. (Hart et al., 1991) In gestational

tissue, IL-4 is produced by decidual cells, fetal membranes, cytotrophoblasts as well as maternal

and fetal endothelial cells. IL-4 expression increases in gestational tissues such as the trophoblast,

decidua and amnion throughout pregnancy (Sykes et al., 2012), suggesting a role in pregnancy

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maintenance. However, IL-4 levels do not correlate to fetal development in mice as IL-4

knockout mice do not show impaired fetal growth or development as compared to wild type

controls (Svensson et al., 2001).


IL-10, is a potent anti-inflammatory cytokine, known to influence the JAK/STAT

pathway and inhibit NF-kB activation, to suppress expression of TNF-a, IL-1, IL-6, and IL-12

by human macrophages (Wang et al., 1995; Riley et al., 1999; Saraiva and O’Garra, 2010).

Expressed by placental villous trophoblasts and decidual immune cells (Warner et al., 2013), IL-

10 is highly produced in 1st and 2nd trimesters, but wanes in the 3rd trimester and prior to labour

(Simpson et al., 1998; Hanna et al., 2000). Although IL-10-/- mice do not demonstrate

impairments to fetal development (Svensson et al., 2001), an increased placental size has been

observed in affected animals (Roberts et al., 2003). These mice also show amplified immune

response to low dose LPS and un-methylated CpG dinucleotides – ligands for TLR4 and TLR9

respectively (Murphy et al., 2005; Thaxton et al., 2009).

1.4.2.3 Interleukin- 13 (IL-13)

IL-13 has close sequence and functional homology with IL-4 (Mak, 2006) to exert anti-

inflammatory responses by host cells. In the gut, IL-13 mediates environments which are

unfavorable for the survival of pathogens by inducing secretions of molecules such as

glycoprotein fromgut epithelial cells which detach and remove microorganisms. (Seyfizadeh et

al., 2015) Additionally, in human amnion-derived WISH cells, IL-13 inhibits cytokine-induced

IL-8 and PGE2 production. (Keelan et al., 1998)

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1.4.3 Colony-Stimulating Factors

M-SCF (CSF1), GM-CSF (CSF2), and G-CSF (CSF3) are multifunctional hematopoietic

cytokines that regulate proliferation, differentiation, and survival of macrophages and

neutrophils. M-CSF in gestation is involved in early stages through receptor interaction in

placental trophoblasts to support fetal implantation and placental development. (Fixe and

Praloran, 1997) Furthermore, increased M-CSF in 1st trimester maternal serum is associated with

trisomy 21 and maternal gestational diabetes, whereas decreased M-CSF has been associated

with trisomy 13, trisomy 18, PTB, and placental insufficiencies. (Eckmann-Scholz et al., 2016)

GM-CSF, while bearing inflammatory capacities to enhance granulocyte and macrophage

proliferation and maturation, is also recognized as an anti-inflammatory agent that can influence

dendritic cell signaling to enhance regulatory T-cell localization and function (Bhattacharya et

al., 2015). In mice, GM-CSF has also shown to exhibit neuroprotective effects against

neurotoxin MPTP, rendering mice less vulnerable to neurodegeneration in Parkinson’s Disease

(Kosloski et al., 2013). In a prospective study, increased GM-CSF expression in cervico-vaginal

fluid prior to 24 weeks gestation was detected in association with shortened cervix and PTB

(Chandiramani et al., 2012). G-CSF, administered to monocytes, exhibits anti-inflammatory

characteristics through suppression of LPS-induced cytokine secretions of IL-1b, TNF-a, IL-12

and IL-18 through JAK/STAT mediated pathways (Boneberg and Hartung, 2002). G-CSF

expression is also increased during labour in cervical tissue (Sennström et al., 2000), and in

laboring human myometrium (Shynlova et al., 2012). Recent in vitro studies have also

demonstrated increased production of GM-CSF and G-CSF in human myometrial cells following

static stretch. (Lee et al., 2014)

11

1.4.4 Chemokines


IL-8 (CXCL8) is a major chemoattractant for neutrophils; it is expressed by leukocytes,

fibroblasts, endothelial cells and epithelial cells in response to TLR stimulation as a crucial

moderator of the innate immune response. IL-8 functions to induce phagocytic activity in

activated neutrophils, and to induce target cell exocytosis to secrete histamines, which further

induces leukocyte migration (Mak, 2006). In pregnancy, IL-8 expression increases with labour in

the cervix, (Sennström et al., 2000) fetal membranes, (Young et al., 2002) and myometrium;

(Elliott et al., 2000) secretion of MMP-8 and neutrophil elastase is induced by cervical

neutrophils. IL-8 administration to amniotic cells in vitro (but not in chorionic cells) induces

MMP-9 expression (Arechavaleta-Velasco et al., 2002). IL-8 expression does not fluctuate in

cervicovaginal fluid between sPTB and term laboring samples amongst asymptomatic women

with cervical insufficiency (Yoo et al., 2017), although increased IL-8 in cervical fluid has been

detected in women with preterm pPROM and intra-amniotic infection (Kacerovsky et al., 2014).

High levels of maternal plasma IL-8 have also been characterized in women with severe pre-

eclampsia and in women who miscarry in the 2nd trimester, (Arikan et al., 2012) indicating an

overall unfavourable physiological function of IL-8 in the maintenance of a healthy pregnancy.

Furthermore, high maternal plasma IL-8 in the 2nd trimester has been associated with increased

offspring risk of developing schizophrenia in adulthood (Zhang et al., 2004).

1.4.4.2 Monocyte Chemoattractant Protein-1 (MCP-1)

MCP-1 (also known as CCL2) is a monocyte chemoattractant secreted by monocytes, endothelial

cells and intrauterine tissues (Mak, 2006). MCP-1 triggers the differentiation of monocytes to

macrophages, and stimulates the expression of pro-inflammatory cytokines (Elliott et al., 2000).

Its expression is increased in term human myometrium in labour as compared to not-in-labour,

12

(Sean Esplin et al., 2005) and in the cervix. In women who deliver preterm without infection,

high levels of MCP-1 have also been detected in the cervix (Törnblom et al., 2005). In the

amniotic fluid, high levels of MCP-1 are found both in the presence and absence of intra-

amniotic infection (Esplin et al., 2005).

1.4.5 Other mediators of labour

Inflammatory pathways and cytokines result in activation and increased production of various

molecules that are specifically essential to uterine activation in the onset of parturition. These

include PGs and their receptors, OXTR, and connexins, which help synchronous contraction of

the myometrium. PGs are lipid moieties that are derived from arachidonic acids and converted

into active forms by COX-1 or COX-2 as PGH2, PGI2, PGE2, PGF2a, or PGD2. Systemically, PGs

can induce vasodilation, bronchodilation or bronchoconstriction, and gastrointestinal smooth

muscle contraction or relaxation. PGs in the cervix induce local MMP-9 production, which

induces cervical remodelling processes. (Ekman-Ordeberg and Dubicke, 2012) In the uterus,

increased mRNA expression of COX-2/PTGS2 is detected at term, in both lower and upper

uterine segments. (Tattersall et al., 2008) Furthermore, exogenous administration of PGE2 and

PGF2a have been shown both in vivo and in vitro to induce myometrial contractions, indicating a

crucial role of PGs in the regulation of parturition. (Olson, 2003) One mechanism by which PGs

induce myometrial contractions along with oxytocin is increased intracellular concentrations of

calcium. Both oxytocin and PGs expressions are stimulated by TNF-a and IL-1b, while IL-6

helps increase the expression of OXTR on cell surfaces. Oxytocin also helps to further increase

the production of PGs, supporting a strong positive-feedback loop.

13

1.5 Characterization of vaginal microbiota in non-pregnant women

Ravel et al. defined five community state types (CST) into which healthy vaginal

microbiomes were classified based on the bacterial composition. (Ravel et al., 2011) Correlated

to this classification were vaginal pH, and Nugent score – a classical diagnostic scoring system

observing the shape of rod-shaped bacteria (large, gram-positive scoring 0-4; small, gram-

variable scoring 0-4; and curved- gram-variable scoring 0-2). A score of 7 or higher from a total

of 10 can indicate bacterial vaginosis (BV), the most common disease of vaginal dysbiosis

presented in women of reproductive age. CST-I, II, III and V, dominated by lactobacillus species

L. crispatus, L. gasseri, L. iners, L. jensenii respectively, maintain relatively healthier states and

have low pH and low Nugent scores, representing low risk for pathogen acquisition. Stability of

vaginal health is associated with the dominance of lactobacillus species, which are gram-positive

bacteria. Contrastingly, women with CST-IV classification have higher pH, higher Nugent

scores, and a variety of anaerobes that inhabit the microbiome with a relatively lower presence of

lactobacillus species. Individuals characterized to CST-IV have increased risk of acquiring

sexually transmitted diseases and gynecological complications such as BV or vaginal

candidiasis. (Romero et al., 2014b)

Although classifying the vaginal microbiome of women help identify those who have an

increased risk of acquiring diseases of vaginal dysbiosis, routine events such as the menstrual

cycle and sexual activity cause changes in vaginal microbiome, promoting fluidity between

CSTs, most commonly between CST-I (L. crispatus) and CST-III (L. iners). (Ravel et al., 2011)

The precise role of these species in contribution to vaginal health has been debated as their

presence has also been detected in CST-IV microbiomes. Recent studies have demonstrated

associations between L. iners and vaginal inflammation and further clinical diagnoses of vaginal

14

dysbiosis syndromes. (Kindinger et al., 2017) Dominance of L. iners has also been associated

with short cervixes and preterm birth <34 weeks. (Kindinger et al., 2017)

To understand such discrepancies and to elucidate the overall functional mechanisms of

lactobacilli, scientists have studied their secreted components. Most widely considered of the

lactobacilli secretion is lactic acid, which maintains a low vaginal pH that is unfavorable for

proliferation of pathogenic species. Lactic acid is found in two isomeric forms – L- and D-lactic

acid. L-lactic acid is expressed by both the vaginal epithelium and commensal lactobacilli, while

D-lactic acid is only produced by bacterial species. However, the amount of D-lactate production

is variable between lactobacilli species, which may explain discrepancies in the protective

functions of different lactobacilli. Studies indicate that women whose microbiomes are

dominated by L. crispatus present higher concentrations of D-lactic acid than those dominated

by L. iners, or pathogenic Gardnerella species, and that L. iners species are incapable of

producing D-lactic acid (Witkin et al., 2013).

1.6 Characterization of vaginal microbiota in pregnant women

Lactobacilli colonization is supported by metabolites of mucosal glycogen, which

increase with high levels of estrogen. (Spear et al., 2014) Thus, lactobacilli are absent from

vaginal flora of pre-pubescent and post-menopausal women, and are more abundant and stable

during pregnancies as compared to non-pregnant states. The vaginal microbiome of pregnant

women demonstrates augmented presence of L. vaginalis, L crispatus, L. gasseri, and L.

gensenii, and lower presence of bacteria commonly associated with CST-IV. However, women

categorized in CST-IV, lack this abundance of lactobacilli and face an increased risk of

complications like BV during pregnancy. While some studies have reported no associations

between the bacterial composition of a vaginal microbiome and the rates of PTB, (Romero et al.,

15

2014c) others have found an association between higher abundance of pathogenic bacteria in

vaginal microbiome of pregnant women with previous history of PTB and who then go on to

deliver preterm (Callahan et al., 2017). Some have indicated that a diagnosis of BV during

pregnancy can increase a woman’s risk of delivering PTB by up to two-fold (Guaschino et al.,

2006). Attempts to treat BV and other complications of vaginal dysbiosis with antibiotics in

pregnant and non-pregnant women, are largely insufficient as they only demonstrate success in

eradicating the pathogens temporarily but fail to prevent its recurrence. Instead, clinical studies

have demonstrated adjunct use of oral lactobacilli probiotics with antibiotics in non-pregnant

women help restore homeostasis of the vaginal microbiome to a more stable state, strengthening

the health of the woman (Anukam et al., 2009). One such study used a combination of two

commensal vaginal lactobacilli species, L. rhamnosus GR-1 and L. reuteri RC-14, which has

been found to persist in the vaginal microbiome for up to 19 days following intra-vaginal

administration, (Gardiner et al., 2002) to improve the cure and recurrence rate of BV (Anukam et

al., 2006).

1.7 Probiotics

Probiotics, which are "live microorganisms which when administered in adequate

amounts, confer a health benefit on the host" (FAO and WHO, 2001), have demonstrated no

adverse effects when used during pregnancy, and are deemed safe and acceptable. (Dugoua et al.,

2009; Lindsay et al., 2014) As such, probiotic Lactobacillus rhamnosus GR-1 supernatant

(GR1SN) was used in a recent in vivo mouse study to show its preventative effect to delay the

onset of LPS-induced PTB (Yang et al., 2014). The same study found reduced expression of

various inflammatory cytokines and chemokines in gestational tissue and peripheral maternal

blood, indicating potential mechanisms behind the phenomenon.

16

1.7.1 Beneficial effects of probiotic administration

To date, most work examining the effects of lactobacillus administration on TLR regulation

has been conducted in context of the gut. In the gut, lactobacilli administration appears to result

in an improved immune stability, and resilience to endotoxin challenge. Examination of TLR

expression and function in Peyer’s patches following oral administration of live L. casei in mice

that also received salmonella challenge show an increased expression of TLR2, 4, and 9 as well

as reduced TNF-α production. (Castillo et al., 2011) Similar effects were also observed when

secondary intestinal epithelial cells (HT-29) were exposed to L. acidophilus in vitro. Cells

treated with the probiotic demonstrated a resistance to salmonella-induced increases in TLR2 and

TLR4 expressions. (Moshiri et al., 2017) Furthermore, a study which examined the effects of

live and heat-killed L. plantarum administration in vitro to secondary intestinal cells (Caco-2)

reported that lactobacilli impaired TLR4 mediated NF-kB signalling pathways, as evidenced by

increased expression of inhibitory molecules of the pathway such as TOLLIP, SOCS1, and

SOCS3. (Chiu et al., 2013) However, such effects remain highly specific for bacterial species

and host cell interaction. In an in vivo mouse study where live bacteria or supernatant of L.

rhamnosus GG were prophylactically administered by oral gavage prior to radiation to induce

apoptosis in crypt cells of the small intestine, a protective effect of lactobacilli was ablated in

MyD88-/-, TLR2-/-, and COX2-/- mice, but unaffected in TLR4-/- which implies mechanisms

that function independent of TLR4. (Ciorba et al., 2012) In a recent study, live bacteria or GR-

1SN has been shown to inhibit hypertrophy in neonatal rat ventricular cardiomyocytes induced

by phenylephrine, an a1-adrenergic receptor agonist, (Ettinger et al., 2017) while L. acidophilus

promoted growth of epithelial and myofibroblast cells in chicken embryo organoids cultured in

vitro. (Pierzchalska et al., 2017) Lactobacilli components such as isolated exopolysaccharides

from L. rhamnosus GG have also been injected into mouse fat pads to show improved lipid

17

metabolism, evident through decreased triacylglyceral accumulation and absence of

inflammation. Interestingly, this effect was abrogated in TLR2-/- mice. (Zhang et al., 2016)

In vitro work examining the role of Lactobacilli in mouse macrophages and human

monocytic cell lines demonstrated an increase in IL-10 expression. (Kim et al., 2006; Martins et

al., 2011) Furthermore, G-CSF expression and secretion have also been shown to increase with

Lactobacilli exposure, which affects immune cells in a paracrine manner to inhibit TNF-a

production. (Kim et al., 2006) L. rhamnosus administration in malnourished mice demonstrating

depleted cytokine production, restored their immune response to control levels, and showed

increased levels of IL-10 production above control levels. (Kolling et al., 2015)

In clinical studies, patients on dialysis treatment receiving oral Lactobacili have

demonstrated increased IL-10 expression, (Wang et al., 2015) potentially offering protection

from common incidences of inflammation observed in dialysis patients.

Despite the extensive amount of work that has been conducted to examine the role of

lactobacilli in the gut, examination of their role in the maintenance of labour remain limited. In

vitro studies examining human placental trophoblast cells pre-treated with GR1SN prior to LPS

stimulus demonstrated TNF-a suppression and increased IL-10 secretions as compared to LPS

control. (Yeganegi et al., 2009, 2011) Chemical inhibition of JAK and p38 revealed the

dependency of GR1SN induced IL-10 secretions on JAK/STAT and MAPK pathways. IL-4

levels were not altered following GR1SN treatment in vitro in human trophoblasts, nor in in vivo

mouse experiments, remaining consistently high between groups with inflammatory insult and

Lactobacilli treatment. (Yeganegi et al., 2009, 2011; Yang et al., 2014) Similar suppression of

LPS-induced cytokine secretions by GR1SN were observed in in vitro decidual cultures. (Li et

al., 2014)

18

Most notably, prophylactic intraperitoneal administration of GR1SN was able to delay

the onset of PTB induced by intrauterine LPS in mice, and was associated with altered cytokine

and chemokine expression in various gestational tissues, including myometrium. (Yang et al.,

2014)

A clinical trial of women receiving oral probiotic supplements (VSL#3), comprised of

Lactobacilli, Bifidobacterium, and Streptococcus strains during late gestation demonstrated an

alteration in the bacterial components detected in vaginal swabs, demonstrating a more stable

microbiota as compared to control patients. IL-4 and IL-10 expression in vaginal samples of

women who took VSL#3 were maintained while a decrease in levels of these cytokines were

observed in control samples (Vitali et al., 2012). In another in vitro experiment, combination

treatment of L. rhamnosus GR-1 and L. reuteri RC-14 exhibited strong antifungal effects against

vulvovaginal candidiasis-causing Candida glabrata isolates, (Chew et al., 2015) suggesting a

protective function of vaginal probiotics to microbial infections.

Overall, it is becoming increasingly more evident that the commensal vaginal bacteria

hold many protective functions in immune regulation by inducing anti-inflammatory responses

from various human tissues, including blood leukocytes, gut epithelia, cervical, placental, and

uterine cells. This immune regulation in gestational tissues is an important target for therapeutic

interventions of infectious-induced sPTL as it is well-established that labouring processes are

highly regulated by inflammatory molecules like cytokines that activate labour-associated

molecules. Although exact mechanisms by which these effects are exerted have yet to be

determined, there is increasing evidence that active compounds within lactobacilli secretions

play a crucial role. Prophylactic use of lactobacilli species and their secreted components may

offer new therapeutic solutions in targeting the immune regulation of infectious PTL.

19

1.8 Rationale and Hypothesis

1.8.1 Rationale

Previous in vivo experiments have demonstrated that prophylactic administration of GR1SN

prior to intrauterine infusion of LPS delayed the onset of PTB in mice and lowered the

expression of inflammatory cytokines in various gestational tissues. Similarly, in vitro

experiments showed the inhibitory effect of GR1SN on cytokine secretion by primary human

decidual, placental cells and leukocytes. The inflammatory response was greatest in the

myometrium following LPS administration, which suggested a crucial role in myometrial

immune regulations for the onset of LPS-induced PTB. The active components within GR1SN

have not been identified, and the mechanisms by which they exhibit cytokine suppression in host

cells are unknown. Through this thesis, I sought to examine the effects of prophylactic treatment

of human myometrial cells by GR1SN for suppression of LPS-induced cytokine secretions.

Examination of such pathways and identification of the active components of GR1SN will offer

clearer understanding of potential molecular targets in the myometrium for preventing PTB in

human.

1.8.2 Hypothesis & Objectives

I hypothesize that GR1SN contains active components that can be used in vitro to

suppress LPS-induced cytokine secretions by myometrial cells.

The two main aims of this study were to (1) examine the in vitro effects of GR1SN on

LPS-induced cytokine secretion by human myometrial cells and (2) investigate the mechanisms

behind the active components of GR1SN in myometrial cells. Additionally, a third aim was

added to (3) test whether prophylactic administration of GR1SN can prevent the onset of PTB

induced through systemic administration of LPS in pregnant CD-1 mice.

20

CHAPTER 2: MATERIALS AND METHODS


2.1 MRS agar and broth preparation

De Man, Rogosa, and Sharpe (MRS) broth was prepared by mixing 55g of MRS powder (BD,

Ontario, Canada) in 1L of double deionized water. For MRS agar plates, 15g of agar power was

added to the broth concoction. Broth and agar were autoclaved for sterility.

2.2 Lactobacilli supernatant (SN) preparation and modification 2.2.1 MRS-based SN (GR1SN-MRS)

Lactobacillus rhamnosus GR-1 was grown anaerobically at 37oC for 48h on MRS agar plates and

streaked for purity. Single colonies were transferred to 10mL of sterile MRS broth and

anaerobically incubated for 12.5h at 37oC to mid-exponential phase at OD620nm ~0.9

(representing ~108-109 cfu/mL of bacteria) and centrifuged at 4000g for 10min at 4oC. The

supernatant (GR1SN) was passed twice through 0.22µm pore filters for removal of residual

bacteria.

2.2.2 PBS-based GR1SN (GR1SN-PBS)

At the time of GR1SN-MRS collection, the bacteria pellet was washed three times in PBS

without calcium and magnesium and incubated in PBS with calcium and magnesium for 2h at

37oC under anaerobic conditions. SN was collected by centrifugation at 4000g for 10min and

filtered twice through a 0.22µm filter to remove residual bacteria.

2.2.3 GR1SN Size Fractionation

Serial size filtration was conducted using Amicon Ultra membrane filtration tubes (EMD

Millipore, Ontario, Canada). 50kDa tubes were filled with GR1SN and centrifuged at 4000g for

10 min, following manufacturer recommendations. Filtrate (< membrane pore size) was collected

21

and was filtered using 10kDa tubes at 4000g for 40 min, and filtrate from 10kDa tubes was

further fractioned through 3kDa tubes at 4000g for 10min, following manufacturer

recommendations. Individual residues (> membrane pore size) were collected and resuspended to

starting volume using original solute.

2.3 Macromolecule Detection & Fractionation in -SN

2.3.1 Protein Detection

Bicinchoninic acid assays (BCA and microBCA, Pierce Biotechnology, Thermo Fisher Scientific,

MA, USA) were used to quantify total protein in GR1SN. Standards were prepared using

manufacturer supplied bovine serum albumin (BSA) for a working range of 20 – 2,000µg/mL for

BCA kits, and 2-40µg/mL for microBCA kits. For BCA kits, 2µL of sample were loaded with

38µL of supplied working reagent into a 96-well plate, which was shaken for 30 seconds using a

plate shaker, and incubated at 37˚C for 30 min. When using microBCA kits, 150µl of sample

were loaded with 150µl of working reagent in a 96-well plate, shaken, and incubated at 37˚C for

2 h. Absorbance readings were conducted using µQuantTM software (BioTek® Instruments, Inc.,

VT, USA) at 562nm following manufacturer recommendations.

2.3.2 Nucleic Acid Detection

Agarose gels were prepared by adding 1.5% (w/v) of agarose powder to 1xTris-acetate-EDTA

(TAE) buffer. Solution was warmed in microwave in three 1 minute cycles to dissolve agarose

powder. 4µl/100mL of RedSafe Nucelic Acid Staining Solution (iNtRON Biotechnology, South

Korea) was added to solution. Solution was left at room temperature for 2-3 minutes to cool, and

poured into mould with appropriate combs for well number and size. Gel was left to polymerize

at room temperature for ~20 minutes. Samples were mixed with 3x DNA loading buffer and

22

loaded into wells. Gel was run at 100V in buffer chambers filled with 1xTAE. Gels were

visualized with a UV imager at 30 second exposure.

2.3.3 Specific elimination of GR1SN macromolecule function

a. Protein: Two different approaches were used to remove functional protein from the

supernatant. GR1SN was (1) heat-treated for 30 minutes at 95˚C to denature all native

proteins, or (2) incubated 1:1 (v/v) in trypsin-EDTA for 20 minutes at 37˚C for protein

degradation: trypsin activity was inhibited by adding DMEM/F-12 media at five times the

volume of trypsin.

b. Nucleic Acid: To degrade nucleic acids in GR1SN, a synthetic nuclease, Benzonase

Nuclease (BN, Sigma-Aldrich, Ontario, Canada), was used. BN targets all forms of DNA

and RNA; single-stranded, double-stranded, and circular moieties. BN has no proteolytic

activity making it safe for use in solutions with protein. Prior to treatment with BN,

GR1SN was diluted 1:1 in a solution of 50mM of Tris-HCl (pH 8.0), 1mM MgCl2, and

0.1mg/mL of BSA (BioShop® Canada, Ontario, Canada) following manufacturer

recommendations, and was incubated in various concentrations of BN at 4ºC for 16h. BN

activity was inhibited using 100mM of guanidine HCl, following manufacturer

recommendations.

2.4 In vitro cell culture

2.4.1 Primary human myometrial tissue culture

Myometrial tissue biopsies were collected from term not-in-labour women undergoing

elective caesarian section at Mount Sinai Hospital after receiving patient’s consent. (n=5)

Primary myometrial cells were prepared by enzymatic digestion and cultured in phenol red-free

DMEM/F-12 media (Gibco) supplemented with 10% FBS (Wisent Bio Products, QC, Canada).

23

At passage 4 confluent myometrial cells were serum starved in DMEM/F-12 + 1% ITS (Gibco).

Cells were stimulated with 100ng/mL of LPS (E. coli 055:B5, Sigma) for 24 hours. Cell-

conditioned media (CM) were collected and analyzed for secreted cytokine concentrations using

a Human 9-plex Cytokine assay (Bio-Rad Laboratories, Ontario, Canada).

2.4.2 hTERT-HM culture

A human myometrial smooth muscle cell line immortalized with human telomerase reverse

transcriptase (hTERT-HM, gift from Dr. J. Condon) was seeded into 6-well plates (100,000

cells/well) in phenol red-free DMEM/F-12 media (Gibco) supplemented with 10% FBS (Wisent)

and 1% Penicillin/Streptomycin/Amphoterin B (Gibco). When ~ 60% confluent, cells were

serum-starved in DMEM/F-12 supplemented with 1% ITS-A (Gibco), and 1% Pen/Strep with

Amphoterin B (Wisent). After 8 hours in serum-free media (SFM) to synchronize cell cycles,

cells were administered a vehicle or pre-treatment with GR1SN for 16 hours, followed by

vehicle or LPS to induce inflammation for 8 hours. CM were collected and analyzed for secreted

cytokine concentrations.

2.4.3 TLR-agonist kit (InVivogen)

A human TLR-agonist kit was used to individually stimulate TLR 1-10. (InVivogen, CA, USA)

Dose-response experiments were conducted by exposing cells to a range of concentrations of

individual agonists for 8 hours following manufacturer’s recommendations. Pam3CSK (TLR1/2

agonist) was tested at 0.01, 0.1, 0.5, and 1.0µg/mL; LPS-EK (TLR4 agonist) was tested at 1, 10,

100, 1000ng/mL; FSL-1 (TLR2/6 agonist) was tested at 1, 10, 100, 1000µg/mL.

24

2.5 Cytokine Quantification Assays

2.5.1 Enzyme Linked Immunosorbent Assay

Sandwich enzyme-linked immunosorbent assays (ELISA) were used to determine specific

cytokine concentrations in CM following various combinations of treatments. Ready-SET-Go! ®

ELISA kits for human IL-6 and IL-8 were purchased from eBioscience (California, USA).

DuoSET® ELISA kits and auxillary kits for human MCP-1, and G-CSF were purchased from

R&D Systems Inc. (MN, USA). Conditioned media were diluted 1:10 for IL-6 and IL-8, and 1:5

for MCP-1 assays using supplied diluent solutions from the manufacturer to ensure the

absorbance reading would remain in range of the standard curve. Absorbance readings were

conducted using µQuantTM software (BioTek® Instruments) at manufacturer-specified

wavelengths.

2.5.2 Human 10-multiplex cytokine assay (Bio-Plex)

Cytokine, chemokine, and growth factor concentrations in CM were determined using a human

10-plex Bio-Plex Multiplex Cytokine Immunoassay (Bio-Rad). The assay measured

concentrations of IL -6, IL-8, MCP-1, TNF-a, IFN-g, IL-2, IL-4, IL-10, G-CSF, and GM-CSF.

Data were analyzed using Bio-Plex Manager (version 5.0, Bio-Rad) and results are presented as

concentrations (pg/mL).

25


2.6 Animals

All animal experiments were approved by the Animal Care Committee of Toronto Centre for

Phenogenomics (Animal Use Protocol 0164H). Guidelines set by the Canadian Council for

Animal Care were strictly followed in handling mice. Animals were housed in a pathogen-free,

humidity controlled 12h light, 12h dark cycle facility with free access to food and water. Female

CD-1 mice were naturally bred; the morning of vaginal plug detection was designated as

gestational day (GD) 1 of full term 19-20 days.

2.7 LPS preparation

Stock LPS (E. coli 055:B5, Sigma) was received at 1mg/mL, which was diluted to in PBS

without calcium or magnesium to working concentration.

2.8 Intraperitoneal injections

Each mouse received 1.5mg/kg of LPS in saline per intraperitoneal injection, which resulted in

low maternal morbidity, while maintaining effectiveness in inducing preterm delivery.

Intraperitoneal GR1SN injections (200µl/30gmouse) were conducted on GD14, followed by

injections of LPS (1.5mg/kgmouse) 24h later, on GD15 and 30minutes – second injection of

GR1SN. Mice were anesthetized through inhaled isoflurane and solutions were injected into the

left upper quadrant of the abdominal cavity. Care was taken to avoid puncture of pups as well as

the femoral artery.

2.9 Animal Groups

(1) Vehicle control: Intraperitoneal injections of MRS broth and saline were administered as

vehicle for GR1SN and LPS respectively. This group assesses whether there are components

within the MRS broth or volume-dependent effects that alter the results.

26

(2) GR1SN control: Intraperitoneal injections of GR1SN on GD14 and 15 were administered in

conjunction with saline (LPS vehicle) on GD15. This group tests for potential effects of GR1SN

to ensure it does not induce PTL.

(3) LPS control: This group received intraperitoneal injections of LPS on GD15 without prior

GR1SN or MRS broth. This was done to confirm the occurrence of PTL as a result of systemic

inflammation and to establish grounds for comparison of the treated group in its benefitting

effects of PTL prevention.

(4) GR1SN + LPS treatment: This treatment group received two intraperitoneal injections of GR-

1SN on GD14 and 15 followed by LPS injections on GD15. All groups were observed at 2hour

intervals for the first 24hours, at 4hour intervals until 48hours, and at 12hour intervals until

72hours. Animals that did not deliver preterm were sacrificed at term (GD18.75).

C. STATISTICAL ANALYSIS

All statistical analysis performed in this study were carried out using PRISM software (version

5). Cytokine concentrations were analyzed with One-way ANOVA followed by Dunnett post-

test comparing to negative or positive controls accordingly. Data are expressed as mean values ±

SEM. P<0.05 was considered statistically significant.

27

CHAPTER 3: RESULTS

A. IN VITRO EXPERIMENTS 3.1 Establishing an appropriate cell line & inflammatory stimulus

To determine myometrial immune response, CM from primary human myometrial cells

stimulated with LPS (100ng/mL) was analyzed using a human BioPlex assay for 9 cytokine

markers of inflammation. Cytokines IL-6, IL-8, and MCP-1 were significantly induced in

response to LPS stimulus (Figure 1a). However, due to large patient-to-patient sample variation,

(Figure 1b) we decided to employ a human myometrial smooth muscle cell line immortalized

with telomerase reverse transcriptase (hTERT-HM) for future experiments. LPS treatment

(100ng/mL) of hTERT-HM cells for 8 hours resulted in significantly induced IL-6, IL-8, and

MCP-1 secretions, (Figure 2) comparable to results of cytokine induction by LPS in primary

myometrial cells, human trophoblasts (Yeganegi et al., 2010), human decidual cells (Li et al.,

2014), and maternal peripheral blood leukocytes. (Meshkibaf et al., 2015) Such similarities

validated the use of hTERT-HM for all future in vitro experiments.

3.2 GR1SN-MRS pre-treatment attenuates LPS-induced inflammatory responses in hTERT-

HM cells

Previous in vivo experiments demonstrated that injections of GR1SN-MRS alone did not alter

gestational length, maternal morbidity, or fetal weight but that prophylactic administration of

GR1SN-MRS prior to local LPS challenge delayed the onset of LPS-induced PTL by 43%.

(Yang et al., 2014) Delayed PTL was found to be associated with significantly reduced

expression of IL-6, TNF-a, CSF-2, IL-3, IL-9, IL-12, IL-13, and IL-17 in the myometrium of

treated mice. To examine potential anti-inflammatory effects of GR1SN-MRS on human

28

myometrium, hTERT-HM cells were pre-treated in vitro with 1, 2, or 5% v/v GR1SN-MRS,

followed by induction of inflammatory response by LPS (100ng/mL),

CM was collected and IL-6, IL-8, or MCP-1 cytokine concentrations were analyzed by

ELISAs. Three important observations were made. First, GR1SN-MRS administration alone for

24 hours significantly induced IL-6 secretion in a dose-dependent manner, resulting in 6.2-fold

increase at 5% v/v compared to hTERT-HM cells treated with SFM (negative control). IL-8 and

MCP-1 secretions were not affected by GR1SN-MRS exposure. Secondly, concurrent

administration of GR1SN-MRS with LPS did not result in suppression of IL-6, IL-8, or MCP-1

secretions relative to LPS (100ng/mL). Thirdly, myometrial cells exposed to low dose of

GR1SN-MRS (1% v/v) for 16 hours prior to LPS administration, secreted significantly lower

levels of IL-8 (54%, P<0.001) and MCP-1 (65.5%, P<0.001) as compared to LPS treatment.

(Figure 3) IL-6 secretions were 36% lower following 1% v/v GR1SN-MRS pre-treatment, but

this was not statistically significant by one-way ANOVA analysis with Dunnett’s Multiple

Comparison post-test.

3.2.1 Identification of macromolecular compounds in GR1SN-MRS

The macromolecular components of GR1SN-MRS that potentially influenced the

secretion of inflammatory cytokines by myometrial hTERT-HM cells were examined. BCA

assays, which quantify total protein in solution, detected 2000µg/mL of protein in MRS broth,

and 4000µg/mL of protein in GR1SN-MRS (Figure 4a) and the presence of nucleic acids were

visualized with a 1.5% agarose gel (Figure 4b). To examine individual contributions of each

macromolecular component, GR1SN-MRS was differentially modified before hTERT-HM pre-

treatment. hTERT-HM cells pre-treated with heat-incubated GR1SN to degrade proteins (‘Heat’,

Figure 5) showed cytokine secretion values similar to non-heat deactivated crude GR1SN-MRS.

(‘1%’, Figure 5)

29

Figure 1. Cytokine secretion by primary human myometrial cells in response to LPS stimulus. Conditioned media from untreated (control) cells and cells treated with 100ng/mL of LPS for 24 hours were analyzed for cytokine concentrations using a Luminex human 9-plex cytokine assay. Secreted cytokine concentrations are shown as absolute concentrations in pg/ml. Individual dots represent different patients (n=5). Statistical significance was determined by individual t-tests (*P<0.05; **P<0.01). This experiment was performed with assistance from Dr. O. Shynlova and A. Dorogin

30

Figure 2. Comparative dose and time responses following LPS administration in hTERT-HM cells. a) Cells were treated with various doses of LPS (0.1-1000 ng/mL, grey to black bars) for 8hours. Dose response values are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Significance was determined by One-way ANOVA followed by Dunnett post-test compared to vehicle control. b) Cells were treated with 100ng/mL of LPS for various time points. Data are shown as absolute concentrations [pg/mL]. Significance was determined by t-tests between vehicle and LPS values at individual time points. (*P<0.05, **P<0.01, ***P<0.001)

a.

b

31

Figure 3. Effect of GR1SN-MRS treatment on hTERT-HM cell cytokine secretion following LPS stimulus. hTERT-HM cells were treated with various doses of GR1SN-MRS (1, 2, 5% v/v, shades of orange) or MRS broth (1% v/v, veh) for 16 hours prior to or concurrently with (1%, v/v, Co-1%, brown bars) LPS (100ng/mL). Cytokine secretions are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Statistical significant difference was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS stimulus (black bars). (*P<0.05; **P<0.01; *** P<0.001) (n=9)

32

However, GR1SN-MRS modified with trypsin lost its ability to prevent cytokine

induction in hTERT-HM cells following LPS stimulus, (‘Trypsin’, Figure 5) as characterized by

high concentrations of secreted IL-6, IL-8, and MPC-1 comparable to levels secreted by hTERT-

HM cells treated with LPS only. (‘LPS+’, Figure 5)

Degradation of nucleic acid in GR1SN-MRS following incubation with BN at 50U/mL

and 100U/mL was verified using a 1.5% agarose gel. (Figure 4d) Pre-treatment with BN-

modified GR1SN-MRS demonstrated similar suppression of LPS-induced cytokine secretions by

hTERT-HM cells as compared to non-BN-treated crude GR1SN-MRS, (‘BN’, “1%”, Figure 5)

which suggests a lack of immune regulatory effect exerted by nucleic acids in GR1SN-MRS.

3.3 MRS broth influences myometrial inflammation

Data presented above indicated the potential role of proteins in GR1SN-MRS on the induction of

IL-6 secretions by hTERT-HM cells. To test the effect of MRS broth alone, various doses of

broth ranging from 0.1, 0.5, 1, 5% v/v were administered to hTERT-HM cells. It was determined

by cytokine analysis that similar to the effects of GR1SN-MRS, IL-6 concentrations were

significantly (P<0.05) induced by MRS alone as compared to the negative control (SFM),

resulting in a 4.5-fold induction following 5% v/v MRS treatment. (Figure 6) A significant

reduction of IL-6 secretion (57.6%, P<0.05) was also observed following 0.1% v/v MRS pre-

treatment prior to LPS stimulus (100ng/mL). Secretion of chemokines IL-8 and MCP-1 were not

induced by MRS broth alone, but MRS pre-treatment at 1%, 2%, 5% v/v resulted in significant

suppression of both cytokines (P<0.001) when administered prior to LPS stimulus. (Figure 6)

33

Figure 4. Characterizing macromolecules in GR1SN-MRS a) Protein concentrations in MRS broth (1941.7µg/mL) and GR1SN-MRS (3946.7µg/mL) were detected using a BCA assay. (n=3) b) 1.5% agarose gel depicting nucleic acid content in GR1SN-MRS. A band of nucleic acid is visible in unmodified GR1SN (white band, SN) but is absent in GR1SN treated with BN (SN+BN). L, Ladder.

b.

34

Figure 5. Relative cytokine secretions by hTERT-HM cells pre-treated with modified GR1SN-MRS prior to LPS stimulus. hTERT-HM cells were pre-treated with non-modified GR1SN-MRS (orange bars), or with GR1SN-MRS modified to degrade nucleic acids (BN, green bars), denature protein structures (Heat, pink bars) or degrade proteins (Trypsin, violet bars) prior to LPS (100ng/mL). Cytokine secretions are shown as fold change (mean ± SEM) relative to untreated cells (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS stimulus (black bars). (*P<0.05; **P<0.01; ***P<0.001)

35

3.4 GR1SN-PBS pre-treatment can suppress LPS-induced cytokine and chemokine secretions

by hTERT-HM cells

Following observation that MRS broth alone exerted immune regulatory effects on

hTERT-HM cells, PBS-based GR1SN was developed as described in Methods. Next, GR1SN-

PBS was administered to hTERT-HM cells before LPS insult (100ng/mL), CM was collected

and analyzed for cytokine secretions by ELISA. GR1SN-PBS treatment alone did not result in

increased secretion of IL-6, IL-8, or MCP-1 as compared to SFM control. Concurrent treatment

of cells with both GR1SN-PBS and LPS (100ng/ml) did not inhibit cytokine secretions by

myometrial cells in response to LPS stimulus. GR1SN-PBS pre-treatment for 18 hours prior to

LPS showed significant (p<0.05) suppression of IL-6 by 47.11%, IL-8 by 34.8% and of MCP-1

by 38.4% as compared to LPS induction. (Figure 7)

3.4.1 Identification of functionally active macromolecules in GR1SN-PBS

BCA analysis of GR1SN-PBS found 22µg/mL of protein in the supernatant, a significant

reduction of total protein content relative to GR1SN-MRS (Figure 8a). GR1SN-PBS was heat-

treated or incubated with trypsin to remove functional protein activity. hTERT-HM pre-treated

with either of heat-treated or trypsin-incubated GR1SN-PBS did not demonstrate suppressed

cytokine secretions following LPS stimulus indicating a functional capacity of GR1SN-PBS

proteins on suppressing LPS-induced cytokine secretions by hTERT-HM cells (Figure 9).

36

Figure 6. Effect of MRS broth on cytokine secretion in hTERT-HM. hTERT-HM cells were treated with various doses of MRS broth (0.1-5% v/v, shades of yellow) with or without LPS (100ng/mL). Cytokine secretions are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS stimulus (black bars). (*P<0.05; **P<0.01; ***P<0.001) (n=4)

37

Figure 7. Effect of GR1SN-PBS treatment on cytokine suppression following LPS stimulus. hTERT-HM cells were treated with various doses of GR1SN-PBS (1, 2, 5% v/v, shades of blue) 16hours prior to or concurrently with (Co-SN, dark blue bars) LPS (100ng/mL). Cytokine secretions are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Statistical significant difference was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS stimulus (black bars). (*P<0.05; **P<0.01; ***P<0.001) (n=9)

38

3.4.2 Size Fractionation of GR1SN-PBS

To isolate the active component of GR1SN-PBS, the supernatant was centrifuged using

filtration tubes at 3 different membrane sizes; 3kDa, 10kDa, 50kDa. The amount of protein

within each fraction was quantified by BCA analysis. From the initial 22µg/mL of total protein,

4.24µg/mL was in the >50kDa fraction, and 13µg/mL was found in the <50kDa fraction. The

filtrate of 10kDa (<10kDa) contained 12µg/mL of protein, and 13.5µg/mL of protein was <3kDa.

Pre-treatment of hTERT-HM cells with the >50kDa fraction resulted in significant suppression

of MCP-1 by 50.4% (P<0.001). Pre-treatment with the <50kDa fraction was effective in

significantly suppressing IL-6 and IL-8 by 52.7% (P<0.05), MCP-1 by 54.6% (P<0.001). Pre-

treatment with the <10kDa fraction did not result in significant IL-6 suppression, though IL-8

secretions were reduced by 50.5% (P<0.01) and MCP-1 secretion was suppressed by 64.8%

(P<0.001). Fractions smaller than 3kDa also retained activity to significantly (P<0.01) suppress

cytokines and chemokines, with IL-6 secretions reduced by 32.3%, IL-8 by 53.5%, and MCP-1

by 50%. Alternatively, serial fractionation steps were also performed where all supernatant were

initially passed through the 50kDa filter, and the filtrate (<50kDa) was then passed through a

10kDa filter, and again through a 3kDa filter. This resulted in protein fractions sized between 10

and 50kDa (3.73µg/mL) and between 3 and 10kDa (6.17µg/mL). Pre-treatment of hTERT-HM

cells with each of the fractions prior to LPS stimulus resulted in significant suppression of IL-6

secretion by the proteins from two fractions sized 3<x<10kDa and >50kDa. IL-8 was

significantly suppressed by hTERT-HM pre-treated with two fractions: <3kDa and 3<x<10kDa.

MCP-1 was significantly suppressed following hTERT-HM pre-treatment with 10<x<50kDa and

>50kDa fractions. (Figure 10)

39

Figure 8. Quantifying proteins in GR1SN-PBS a) Protein concentrations in GR1SN-MRS (3946.7µg/mL) and GR1SN-PBS (22.6µg/mL) were detected using a BCA assay. (n=3)

40

Figure 9. Effect of modified GR1SN-PBS on LPS-induced cytokine secretions. hTERT-HM cells were treated with non-modified GR1SN-PBS (blue bars), or with GR1SN-PBS modified to denature protein structures (Heat, green bars) or degrade proteins (Trypsin, violet bars), with or without LPS stimulus (100ng/mL). Cytokine secretions are shown as fold change (mean ± SEM) relative to untreated cells (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS control (black bars). (*P<0.05; **P<0.01; ***P<0.001) (n=4)

41

Figure 10. Effect of hTERT-HM pre-treatment with various GR1SN-PBS size fractions on LPS-induced cytokine secretions. hTERT-HM cells were pre-treated with non-modified GR1SN-PBS (blue bars), or with size-fractioned GR1SN-PBS prior to LPS stimulus (100ng/mL). GR1SN-PBS was separated into fractions between 3 and 10kDa ([3,10kDa], blue horizontal stripes), between 10 and 50kDa ([10,50kDa], blue vertical stripes), or greater than 50kDa (>50kDa, blue diagonal stripes). Cytokine secretions are shown as fold change (mean ± SEM) relative to untreated cells (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS control (black bars). (*P<0.05; **P<0.01; ***P<0.001) (n=3)

42

3.5 Low-dose TLR agonist pre-treatment attenuates LPS-induced inflammatory responses in

hTERT-HM cells.

As described above, concurrent incubation of neither GR1SN-MRS nor GR1SN-PBS

with LPS led to suppression of LPS-induced cytokine secretion by hTERT-HM, while GR1SN

pre-treatment of GR1SN-PBS for 16 hours inhibited LPS-induced inflammatory responses.

(Figure 3, Figure 7) Such findings led me to hypothesize that the active components within

GR1SN do not function by direct effect to inhibit LPS stimulus, but rather offer immune

protection through cell-mediated pathways to suppress LPS-induced cytokine secretion.

Previous studies have demonstrated that GR1SN-MRS induced secretions of anti-

inflammatory cytokines such as IL-4, IL-10 (Yeganegi et al., 2010; Yang et al., 2014) or growth

factor G-CSF, (Kim et al., 2006; Yeganegi et al., 2011; Meshkibaf et al., 2015) to regulate the

overall ratio of pro- to anti-inflammatory state in treated cells. However, it was found using a 10-

plex human cytokine multiplex assay (BioPlex), that hTERT-HM cells do not secrete detectable

levels of anti-inflammatory cytokines or growth factors following GR1SN treatment (Table 1).

Alternatively, mechanisms of endotoxin tolerance also result in cytokine suppression

through cell mediated pathways without influencing secretion of anti-inflammatory cytokines.

Tolerance results through repeated stimulus from same or similar agonists. It has been previously

reported that TLR4, a known receptor for LPS, can become desensitized to repeated LPS

stimulus (homo-desensitization) (Lehner et al., 2001; Dobrovolskaia et al., 2003a) or to repeated

stimulus of other TLRs that share similar downstream inflammatory pathways, such as TLR2

(hetero-desensitization). (Lehner et al., 2001; Dobrovolskaia et al., 2003a) Thus, I hypothesized

that GR1SN-mediated activation of TLRs in hTERT-HM cells during the pre-treatment period

may elicit tolerance to a subsequent TLR4 stimulation by LPS.

43

Table 1. 10-plex BioPlex cytokine assay of media conditioned by hTERT-HM cells treated with GR1SN-MRS. Secreted cytokine concentrations in CM were detected from cells untreated (basal levels), exposed to GR1SN (GR1SN-induced), or LPS-induced (LPS-induced). Average concentrations from 5 experiments and their range are shown in pg/mL. OOR<, values below lowest detectable concentration set by standards.

Cytokine

Lowest detectable

concentration

[pg/mL]

Basal levels

Average (min-max)

[pg/ml]

GR1SN-induced

Average (min-max)

[pg/ml]

LPS-induced

Average (min-max)

[pg/ml]

IL-6 1.41 159.2 (91.3-327.4)

135.1 (95.5-252.5)

827 (424-1293)

IL-8 1.41 565.4 (150.1-1867.1)

1177.7 (127.1-4807.3)

2044 (1152-3330)

MCP-1 3.36 199.1 (136.9-282.7)

169.9 (135.7-219.8)

664 (548-911)

IFN-g 0.91 OOR< OOR< OOR<

TNF-a 3.18 OOR< OOR< OOR<

IL-10 3.39 OOR< OOR< OOR<



G-CSF 1.73 OOR< OOR< OOR<

GM-CSF 0.66 OOR< OOR< OOR<

44

3.5.1 Low-dose LPS and Pam3CSK pre-treatment can suppress LPS-induced cytokine secretion

To test this hypothesis, I first established if hTERT-HM cells could gain tolerance to

repeated stimuli of the same agonist (homo-desensitization). Low-dose LPS (0.01, 0.1,

1.0ng/mL) was used to pre-treat hTERT-HM cells for 16 hours followed by a single high dose of

LPS (100ng/mL) to induce inflammatory signalling. Conditioned media from treated cells were

analyzed by ELISA for inflammatory cytokines IL-6, IL-8, and MCP-1. I found that hTERT-HM

pre-treatment with 0.1ng/mL of LPS prior to 100ng/mL of LPS suppressed secretions of IL-6 by

43.5% (P<0.05), IL-8 by 38.9% (P<0.05), and MCP-1 by 31% (P<0.01) (Figure 11).

Confirmation that TLR4-induced inflammatory responses to LPS stimulus is reduced in

myometrial cells led to further experiments to determine if other TLR agonists were capable of

suppressing LPS-induced cytokine secretion. Manufacturer-recommended doses of functional

Pam3CSK (TLR1/2 agonist) at 0.01, 0.1, 0.5, and 1.0µg/mL and for FSL-1 (TLR2/6 agonist) at

1.0, 10, or 100µg/mL were used to treat hTERT-HM cells for 8 hours. I found that Pam3CSK-

induced secretion of cytokines in a dose dependent manner; 0.5µg/mL of Pam3CSK induced IL-

6 secretions by 3.7-fold (P<0.001), IL-8 by 2.9-fold (P<0.01), and MCP-1 by 2.7-fold (P<0.01),

(Figure 12) whereas FSL-1 was not able to induce any inflammatory cytokine secretions by

hTERT-HM cells. (Figure 13) Using low dose Pam3CSK (0.01µg/mL) which did not induce IL-

6, IL-8, and MCP-1 secretions as compared to SFM control, hTERT-HM cells were pre-treated

for 16 hours prior to a single high dose LPS (100ng/mL). ELISA analysis of CM demonstrated

suppression of LPS-induced pro-inflammatory cytokines IL-6 by 38% (P<0.01), IL-8 by 41%

(P<0.05), and MCP-1 by 43% (P<0.05) as compared to LPS stimulus. (Figure 14)

45

Figure 11. Effect of low-dose LPS pre-treatment prior to high-dose LPS stimulus on cytokine secretions by hTERT-HM cells. Cells were pre-treated with 0.1µg/mL of LPS with (checkered) or without a subsequent LPS stimulus (100ng/mL). Secreted cytokine levels are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS stimulus (black bars). *P<0.05; **P<0.01; ***P<0.001. (n=5)

46

Figure 12. Effect of TLR1/2 agonist (Pam3CSK) dose on cytokine secretion by hTERT-HM cells. Cells were treated with various doses of Pam3CSK (P3K, 0.01-1 µg/mL, violet bars) for 8 hours. Secreted cytokine levels are shown as fold change (mean ± SEM) relative to untreated vehicles (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test. **P<0.01; ***P<0.001. (n=5)

47

Figure 13. Effect of TLR2/6 agonist (FSL-1) dose on cytokine secretion by hTERT-HM cells. Cells were treated with various doses of FSL-1 (1-100 µg/mL, pink bars) for 8 hours. Secreted cytokine levels are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test. **P<0.01; ***P<0.001. (n=5)

48

Figure 14. Effect of low-dose Pam3CSK pre-treatment prior to high-dose LPS stimulus on cytokine secretions by hTERT-HM cells. Cells were pre-treated with 0.01µg/mL of Pam3CSK (P3K) with (checkered) or without LPS stimulus (100ng/mL). Secreted cytokine levels are shown as fold change (mean ± SEM) relative to untreated vehicle (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test relative to LPS stimulus (black bars). *P<0.05; **P<0.01; ***P<0.001. (n=5)

49

3.6 Cytokine regulation in myometrial cells by GR1SN is unique as compared to four other

non-L. rhamnosus GR-1 lactobacilli species.

Since TLR2 is not exclusive to L. rhamnosus GR-1 but is common to bacterial

lipoproteins such as LTA, I examined if non-GR-1 lactobacilli strains would also demonstrate

similar inhibitory effects on cytokine secretion. For this experiment, I used 4 other strains of

probiotic lactobacilli, L. rhamnosus GG, L. lactis, L. casei, and L. reuteri RC-14 provided by Dr.

Sung Kim (Western University, London, Ontario).

All strains were cultured in MRS broth (as described above for GR-1) and their PBS-

based supernatants were used to pre-treat hTERT-HM cells in the same manner as was described

for GR1SN. ELISA analysis of CM from hTERT-HM cells pre-treated with different

lactobacilli-SNs demonstrated that SN from L. rhamnosus GG, lactis, casei, and reuteri RC-14

were not able to suppress LPS-induced cytokine secretions, and that GR1SN-PBS is a unique

suppressor of LPS-induced cytokine secretion by hTERT-HM cells (Figure 15).

50

Figure 15. Effect of five different lactobacilli strains on cytokine suppression in hTERT-HM cells stimulated with LPS. Cells were pre-treated with various doses of distinct Lactobacilli species (blue bars) with (checkered) or without LPS stimulus (100ng/mL). Results are demonstrated as fold change (mean ± SEM) compared to untreated vehicle control (white bars). Statistical significance was determined by One-way ANOVA followed by Dunnett’s post-test compared to LPS stimulus (black bars). *P<0.05; **P<0.01; ***P<0.001. GG, L rhamnosus GG; Lactis, L. lactis; Casei, L. casei; RC-14, L. reuteri RC-14. (n=9)

51


3.7 In vivo effects of GR1SN-MRS pre-treatment to delay the onset of LPS-induced PTL

Previous studies have reported the successful role of prophylactic administration of systemic

GR1SN-MRS to delay the onset of local LPS-induced PTB in CD-1 mice. In the current study, I

examined whether the administration of systemic GR1SN-MRS can also delay PTL induced by

systemic low dose infection. Timed pregnant animals were divided into 4 groups: 1) MRS broth

2) GR1SN-MRS control 3) LPS and 4) GR1SN-MRS + LPS. Deliveries made during the first

24h post-LPS injection were classified as preterm.

Group 1: MRS broth had no effect on inducing PTL. Intraperitoneal injection of MRS broth

(200µL) on GD14 and again on GD15 immediately followed by saline injections (100µL)

showed no indication of preterm labour in treated mice (n=5). All mice were healthy and active

until they were sacrificed at GD18.75. No fetuses were resorbed.

Group 2: GR1SN-MRS does not cause maternal mortality nor induce PTL. Intraperitoneal

injection of GR1SN-MRS (200µL) on GD14 and again on GD15 immediately followed by saline

injections (100µL) showed no indication of preterm labour in treated mice (n=5). All mice were

healthy and active until sacrificed at GD18.75. No fetuses were resorbed.

Group 3: Systemic LPS injections will induce PTL. Intraperitoneal injections of LPS (1.5mg/kg,

100µl) on GD15 resulted in 70% of treated mice (n=10) delivering preterm. Despite the

definition of preterm birth as delivery within 48 hours of LPS injection, it was observed that all 7

mice that delivered preterm did so within 24 hours of LPS administration.

Group 4: GR-1SN-MRS + LPS Treatment. Intraperitoneal injections of GR1SN on GD14 and

again on GD15 immediately followed by LPS injections resulted in PTB in 7 of 12 treated mice

(58%). All mice appeared healthy, active and nesting. There were 6 mice that underwent partial

52

delivery, where only a portion of the uterine horn was emptied. These were classified as having

delivered preterm, and are included in the 58%. All mice that responded and delivered at term

did so at GD18.75. The LPS group was compared to the prophylactic GR1SN treatment group

using Fisher’s exact test. It was determined that the change in rate of PTB due to prophylactic

GR1SN was not significant (p=0.67) as compared to LPS control. (Table 2)

Table 2. The effect of prophylactic GR1SN-MRS on delivery rates of LPS-induced preterm birth in pregnant CD-1 mice

3.8 In vivo effects of GR1SN-PBS pre-treatment to inhibit LPS-induced PTL

Similarly, mice received intraperitoneal injections of GR1SN-PBS prior to intraperitoneal

injections of LPS to determine if GR1SN-PBS can delay PTB induced by systemic LPS

administration. Timed pregnant mice were divided into 4 groups: 1) saline control, 2) GR1SN-

PBS, 3) LPS (1.5mg/kg), and 4) GR1SN-PBS + LPS (1.5mg/kg). Deliveries made during the

first 24h post-LPS injection were classified as preterm.

Group 1: Saline had no effect on inducing PTL Intraperitoneal injection of PBS (300µL) on

GD14 and again on GD15 immediately followed by saline injections (200µL) showed no

indication of PTL in treated mice (n=4). All mice were healthy and active until they were

sacrificed at GD18.5.

Group 2: PBS-based GR-1SN did not cause maternal mortality nor induce PTL. Intraperitoneal

injection of GR-1SN (300µL) on GD14 and again on GD15 immediately followed by saline

Treatment n Preterm Delivery Rate (%) Vehicle 5 0

GR1SN-MRS 5 0 LPS (1.5mg/kg) 10 70

GR1SN-MRS + LPS 12 58

53

injections (200µL) showed no indication of preterm labour in treated mice (n=7). All mice were

healthy and active until sacrificed at GD18.5. No fetuses were resorbed.

(3) Systemic LPS injections induced PTL: Intraperitoneal injections of LPS (1.5mg/kg, 200µl)

on GD15 resulted in 100% of treated mice (n=10) to go into deliver preterm within 24 hours of

LPS administration, indicating “treatment-to-delivery interval” of 22 hours. (Table 3)

(4) GR-1SN + LPS Treatment: Pregnant mice (n=10) received intraperitoneal injections of

GR1SN (300µL) on GD14 and again on GD15 immediately followed by LPS injections (2

mg/kg in 200µl) which resulted in 80% PTB. We did not observe any significant effect of

prophylactic GR1SN-PBS treatment in preventing LPS-induced PTB in pregnant CD-1 mice by

performing a Fisher’s exact test to compare the LPS group with the GR1SN treatment group

(p=0.47).

Table 3. The effect of prophylactic GR1SN-PBS on delivery rates of LPS-induced preterm birth in pregnant CD-1 mice.

Treatment n Preterm Delivery Rate (%) Vehicle 4 0

GR1SN-PBS 7 0 LPS (1.5mg/kg) 10 100

GR1SN-PBS + LPS 10 80

54

CHAPTER 4: DISCUSSION

4.1 In vitro effects of GR1SN pre-treatment on cytokine suppression in hTERT-HM cells stimulated with LPS.

Through my in vitro studies, I have (1) demonstrated the retained suppressive effect of

the secreted compounds of Lactobacillus rhamnosus GR-1 in a PBS-based solution and

introduced endotoxin tolerance as a potential mechanism of action, and (2) observed an effect of

MRS broth alone to influence inflammatory signals in hTERT-HM cells.

4.1.1 Endotoxin Tolerance in hTERT-HM cells

Alternatively, previous in vivo experiments also demonstrated that concurrent

administration of GR1SN-MRS and LPS did not delay the onset of PTB in pregnant mice. These

findings are further supported in the current in vitro study, as concurrent treatment of cells with

GR1SN (in either form, MRS or PBS) and LPS does not suppress LPS-induced secretions of

cytokines and chemokines. In both in vivo and in vitro settings, preventative effects of GR1SN

were only shown when GR1SN was used as a pre-treatment, which suggests the need of a lag

period necessary for GR1SN to induce intracellular changes in host cells, altering host responses

to subsequent LPS stimulation. Studies of GR1SN in other tissue types, such as monocytes,

(Meshkibaf et al., 2015) decidual (Li et al., 2014) and trophoblast cells, (Yeganegi et al., 2009,

2010, 2011) have demonstrated the secretion of anti-inflammatory cytokines such as IL-10 to

explain the effects observed by GR1SN pre-treatment. However, neither primary human

myometrial cells, nor hTERT-HM cells used in the current study secreted detectable amounts of

anti-inflammatory cytokines following GR1SN treatment.

Rather, studies of endotoxin tolerance in human and murine immune cells suggest that

reduced expression of pro-inflammatory cytokines such as TNF-a, IL-1b, and IL-6 is not

55

necessarily paired with induction of anti-inflammatory cytokines like IL-10. (Sato et al., 2000)

“Endotoxin tolerance” refers to a mechanism by which cells become less responsive to a

secondary endotoxin (i.e. LPS stimulus) following pre-treatment with low-doses of identical or

similar agonists that activate overlapping downstream pathways. Tolerance has been well-

established in immune cells by pre-treatments with agonists for TLR2, (Sato et al., 2000) TLR4,

(Dobrovolskaia et al., 2003b) and TLR9 (Cowdery et al., 1996; Gao et al., 1999). A recent study

elucidated the effects of endotoxin tolerance induced by Lactobacillus plantarum treatment in

human intestinal cells (Caco-2), characterized by increased expression of regulatory molecules of

immune activation such as TOLLIP, SOCS1, SOCS3 and their associated suppression of pro-

inflammatory pathways. (Chiu et al., 2013)

I have demonstrated here that GR1SN pre-treatment suppresses LPS-induced cytokine

secretions by hTERT-HM cells. As an initial proof-of-principle experiment, I successfully

induced endotoxin tolerance in hTERT-HM cells by using sub-stimulatory doses of LPS prior to

a high dose simulation with LPS. This resulted in significant suppression of IL-6, IL-8 and MCP-

1 as compared to cells treated with a single high dose of LPS. Subsequent experiments to

identify the specificity of the pathway were performed as endotoxin tolerance can be induced

through TLR2 and TLR4 by bacterial proteins, and through TLR9 by bacterial nucleic acids.

Nucleic acids were identified in GR1SN through nucleic acid staining in agarose gels. However,

it was found that tolerance to LPS in hTERT-HM cells is not mediated through TLR9 activation

by nucleic acids in GR1SN as GR1SN modification with BN incubation to degrade nucleic acid

maintained its effect to suppress cytokine and chemokine secretions. Thus, I assume that the

active component of SN to be of protein/lipoprotein origin. BCA assays of GR1SN indicated

high protein content, which can include lipoprotein moieties secreted or shed by gram-positive

56

bacteria that activate TLR2 receptors and confer tolerance to subsequent endotoxin stimulus.

TLR2 functions as a heterodimer with TLR1 to detect triacylated lipoproteins or with TLR6 to

recognize diacylated lipoproteins. In current studies, I employed highly specific synthetic

agonists for TLR1/2 (Pam3CSK) and TLR2/6 (FSL-1) to individually pre-treat hTERT-HM cells

prior to LPS stimulus. It was found through preliminary dose response experiments that hTERT-

HM cells do not respond to FSL-1, suggesting that TLR2/6 activation is not a plausible pathway

of endotoxin tolerance in hTERT-HM cells. Conversely, Pam3CSK experiments demonstrated a

dose-dependent increase in secretions of pro-inflammatory cytokines by hTERT-HM cells. Pre-

treatment of cells with non-stimulatory doses of Pam3CSKwas able to significantly suppress

LPS-induced secretions of IL-6, IL-8, and MCP-1, indicating a potential role of the TLR1/2

complex in the mediation of cytokine suppression by GR1SN pre-treatment in hTERT-HM cells.

Currently, the exact agonists for TLR2 within GR1SN have not been identified.

However, recent studies examining GR1SN exposure to macrophages have identified heat-labile,

protein-like components between 30-100kDa to promote G-CSF production through TLR2-

dependent mechanisms (Meshkibaf et al., 2015). In the current study, nucleic acid and protein

components were identified in GR1SN. Modifications to remove functional protein activity by

heat and trypsin incubation effectively ablated GR1SN effect to suppress secretion of LPS-

induced pro-inflammatory factors in hTERT-HM cells. Contrastingly, nucleic acid degradation

by incubation with BN did not alter GR1SN effect, although the visible band of nucleic acid was

removed in the agarose gel. As BN was claimed by the manufacturer to degrade all DNA and

RNA, these findings suggested that GR1SN effect can be attributed to protein-like components

and not nucleic acids. However, a study examining the effect of ribonucleases, including BN, on

RNA stability demonstrated that although BN can reduce the amount of intact mRNA by

57

approximately 50%, the amount of intact miRNA was unaffected and remained around 100%.

(Aryani and Denecke, 2015). This suggested that despite the lack of a visible band of nucleic

acid on the agarose gel, there may be unaffected forms of nucleic acid such as miRNA that

persist in the GR1SN to affect inflammatory signalling in exposed cells. Furthermore, pre-

treatment of hTERT-HM cells with sized fractions of GR1SN-PBS demonstrated uncertain

results whereby pre-treatment with fractions greater than 50kDa significantly suppressed IL-6

and MPC-1 secretion to levels comparable to unfractionated (crude) GR1SN-PBS, while IL-8

secretion was not affected.

Lipoprotein sizes vary vastly according to the species from which they are produced and

their endogenous functions, which include regulation of host immune response, bacterial

virulence, and nutrient acquisition for the bacteria to better survive the host. (Nguyen and Götz,

2016) Lipoproteins are generally produced as pre-pro-lipoproteins, and are matured through

lipidation to their functional form. In gram-positive species, they are secreted in extracellular

vesicles which then bind to host receptors, or are found anchored on lactobacilli cell wall.

(Lebeer et al., 2008) For instance, it was determined that the genome of Lactobacillus johnsonii

contains sequences for two lipoproteins, one which may activate CD4+ T-cells and another

which holds similar composition to saliva-binding proteins found in S. sanguis. (Pridmore et al.,

2004) However, their protein function has not been studied. It is also possible that tissue

specificity plays an important role in identifying the functional bacterial lipoprotein.

Alternatively, mechanisms behind endotoxin tolerance can be studied by observing

changes in downstream mediators. Endotoxin tolerance has been shown to be established

through distinct mechanisms which vary depending on the initial TLR and agonist pair that

prime the cells. TLR4-induced endotoxin tolerance operates through mechanisms different from

58

TLR2-induced tolerance. TLR4-mediated endotoxin tolerance leads to decreased surface

expression of the TLR4/MD2 complex, (Nomura et al., 2000; Sato et al., 2000) as well as

decreased expression of adaptor proteins MHP-II, and CD86 (Wolk et al., 2000) at the cell

membrane, without inducing IL-10 secretions in murine macrophages. Monocyte-line human

THP-1 cells tolerized by LPS also show alteration in the NF-kB pathway at downstream sites,

reducing activation of ERK1/2 and p38. (Kraatz et al., 1999) However, TLR2-mediated TLR4

desensitization does not result in reduced TLR4/MD2 expression at the cell surface of murine

macrophages, but can impair NF-kB dimer formations and JNK activation, thus leading to

reduced pro-inflammatory cytokine secretions. (Sato et al., 2000) Moreover, when two different

agonists of TLR1/2 (hBD-3 and Pam3CSK), were used to treat human monocytes, it was

observed that while both stimuli increased IL-6, IL-8, IL-1b and activated MAPK, increased

expression of IL-10 was only observed in cells treated with Pam3CSK and not with hBD-3,

further indicating differential cellular responses to agonists. This was associated with a decrease

in CD86, and was suggested to be due to selective activation of NF-kB in Pam3CSK-treated

cells but not hBD-3. (Funderburg et al., 2011) Endotoxin tolerance can also be induced through

GPCR activation in association with Gi protein activation, which can suppress LPS-induced NF-

kB signaling through increased PI3K and MAPK-phosphatase expression in murine-derived

peripheral macrophages. (Fan et al., 2008) As such, the differential influences exerted by distinct

priming agonists offer targets which can be explored in future experiments to elucidate the

pathways and TLRs that are involved in GR1SN-mediated endotoxin tolerance in hTERT-HM

cells.

A potential concern behind endotoxin tolerance may be that suppression of cytokines and

chemokines may result in persistent bacterial infection as the normal function of cytokines is to

59

activate the immune system in response to infection of foreign organisms. In gestation, persistent

infection may lead to more detrimental consequences to the fetus than PTB. However, endotoxin

tolerance is a complex pathway which involves more than a global suppression of inflammatory

cytokines. In vivo experiments of endotoxin tolerance demonstrated that despite reduced

cytokine expression, mice were more resistant to gram-negative bacterial infection, (Leon et al.,

1992) and a reduced fungal burden following inoculation with Cryptococcus neoformans in

tolerized animals, (Rayhane et al., 2000) indicating an active immune system to fight off

pathogens. Most of the research on endotoxin tolerance has been performed in immune cells

(human and murine monocytes and macrophages) which consistently show attenuated TNF-a

expression, but vast inconsistencies persist in the regulation of many other inflammatory

markers. (West and Heagy, 2002) These discrepancies have been attributed to LPS purity, type

or dose, as well as host cell properties. Indeed, paradoxically, recent studies examining the role

of L. rhamnosus GR-1 treatment in bladder cells have shown that probiotic exposure leads to an

augmented inflammatory response. (Karlsson et al., 2012)

My work has examined the effects of endotoxin tolerance in human myometrial cells

(hTERT-HM), in which TNF-a was produced below detectable levels (lowest detectable

concentration: 3.18pg/mL) both at baseline and following LPS stimulus; and GR1SN-PBS pre-

treatment resulted in suppressed secretion of all IL-6, IL-8, and MCP-1. The novelty of my work

lies in the identification of endotoxin tolerance as a potential mechanism underlying the anti-

inflammatory effects observed in hTERT-HM by GR1SN pre-treatment. I have shown that

uterine myocytes are sensitive to endotoxin tolerance, a mechanism previously limited to studies

in immune and epithelial cells. This further emphasizes the role of the myometrium in

maintaining immune homeostasis throughout gestation, and in regulating the onset of labour in

60

cases preceded by infection. However, it is important to note that probiotic function is highly

tissue-specific: I demonstrated that four other lactobacilli species (L. rhamnosus GG, L. lactis, L.

casei, and L. reuteri RC-14) did not significantly suppress LPS-induced cytokine secretions in

hTERT-HM cells despite their well-established anti-inflammatory effects in other tissue types.

4.1.2 Effect of MRS broth on inflammatory signals in hTERT-HM cells

Interestingly, it was observed that treatment with MRS broth alone can influence

inflammatory signals in hTERT-HM cells such suppression of LPS-induced pro-inflammatory

factors and an increase in IL-6 secretions, similar to the effects of GR1SN-MRS. MRS broth is a

concoction of various factors optimized for the growth of lactobacilli species, including

approximately 2000µg/mL of proteins as was detected by a BCA assay in the current study. Such

factors include beef & yeast extracts, of which absolute components are not disclosed by the

manufacturer (BD Bioscience Inc). Other components include factors that help selective

proliferation of lactobacilli strains such as polysorbate 80, sodium acetate, and magnesium

sulphate. These molecules have been individually studied in various systems on immune

regulation. Polysorbate 80 (PS80) is a surfactant and emulsifier, ubiquitously used in food

preparation, medical, and laboratory settings. Its exposure to reconstructed human epidermal

cells had resulted in increased expression and secretion of IL-1a and reduced cell viability, but

no differences in IL-8 expression. (Coquette et al., 1999) More recently, PS80 administered to

mice at low concentrations was sufficient to induce low-grade inflammation and obesity-related

metabolic syndromes as compared to untreated controls. (Chassaing et al., 2015) Analysis of

male rats treated with PS80 demonstrated elevated levels of IL-6, IL-1, TNF-a, and IFN-g in

liver hepatocytes. (Al-thamir et al., 2013) Similarly, sodium acetate which is also present in

MRS broth, induced secretion of various inflammatory cytokines such as IL-1b, IL-8, TNF-a,

61

and IL-6 in human gastric adenocarcinoma epithelial cells, independent of changes in cell

viability and proliferation. (Sun et al., 2005) Magnesium sulfate (MgSO4), which helps the

growth of lactobacilli, has been vastly studied in vitro to decrease inflammation, and is

commonly used in clinical obstetric practice as a tocolytic for PTB as well as a treatment for

women with preeclampsia to prevent seizures. Preeclamptic placentae also secrete high levels of

TNF-a and IL-6, which can be suppressed by MgSO4 in both humans and mice. MgSO4 thus,

has also been associated with neuroprotective abilities to the developing fetus. (Sugimoto et al.,

2012; Chen et al., 2015) Furthermore, when myometrial tissues from term women undergoing

elective cesaerian sections were exposed to MgSO4, an elevated contractility and increased

intracellular calcium concentrations was detected (Fomin et al., 2006).

The increase in IL-6 secretions by myometrial cells in an in vivo intrauterine environment

can be concerning for fetal development. Rat fetuses are most susceptible to maternal IL-6

during mid-gestation, (Dahlgren et al., 2006) and abnormally high concentrations of IL-6 can

cause hypertension, insulin resistance, and dysregulation of the HPA-axis in adult offspring, as

well as adverse outcomes in fetal neurodevelopment. (Dahlgren et al., 2001; Samuelsson et al.,

2004) High maternal IL-6 in pregnant women has recently been associated with larger right

amygdala volume and higher amygdala interaction with various brain regions in offspring, which

were together associated with reduced impulse control by infants at 24 months. (Graham et al.,

2017) In rodents, increased IL-6 due to maternal immune activation has also been identified as a

key mediator in behavioural, histological, and genetic abnormalities resembling schizophrenia

and autism, as mice with IL-6-/- mice or mice treated with anti-IL-6 antibody did not

demonstrate these alterations. (Smith et al., 2007) Moreover, increased maternal IL-6

concentrations have been associated with increased risk of cerebral palsy, (Yoon et al., 2003)

62

increased fetal adiposity, (Radaelli et al., 2006) and altered neurodevelopmental structures and

hormonal axes. (Dahlgren et al., 2006) In the current study, IL-6 secretions were induced by

GR1SN-MRS alone, which highlights concerns for the developing fetus in a in vivo environment

that should be considered when translating these studies to in vivo or clinical settings. Current

knowledge regarding offspring outcomes from in vivo administration of lactobacilli cultures (live

or supernatant) is limited to fetus viability, birth and placental weight data, and has not extended

to examination of neural deficits, which may be an important factor to consider in future in vivo

studies examining the effects of lactobacilli during gestation. Contrastingly, a recent mouse study

examining the in vivo effects of systemic GR1SN-MRS administration followed by local LPS

stimulation detected a significant suppression of IL-6 and other cytokines in the myometrium,

placenta, amniotic fluid, and maternal plasma, (Yang et al., 2014) demonstrating differential

effects of GR1SN-MRS dependent on the mode of administration. In vitro macrophage studies

demonstrate that anti-inflammatory molecules such as IL-10 and G-CSF are produced in

response to GR1SN-MRS treatment. (Kim et al., 2006) This may suggest a potential mechanism

by which systemic GR1SN-MRS exhibits differential cytokine expression in local gestational

tissues. For instance, when GR1SN-MRS is administered systemically in vivo, increased anti-

inflammatory cytokine secretion by immune cells may regulate local immune states in the uterus.

4.2 In vivo functional discrepancies of GR1SN effect

Contradictory to my original hypothesis, I found that prophylactic administration of IP-

GR1SN was not able to prevent the onset of PTB induced by systemic LPS in pregnant CD-1

mice. Here, I suggest three potential explanations: (1) the mode of LPS administration between

local IU and systemic IP infusions, (2) the tissue specificity of GR1SN, and (3) the composition

of GR1SN-PBS as compared to GR1SN-MRS.

63

4.2.1 Intrauterine vs. intraperitoneal mode of LPS infusion

Previously, it was observed that IP injections of GR1SN-MRS resulted in 43% reduction of PTL

induced by IU-LPS administration (Yang et al., 2014). However, in the present study, IP

administration of neither GR1SN-MRS nor GR1SN-PBS inhibited PTB induced by IP-LPS

(Table 4).

Table 4. Summary of current and previous in vivo results showing rates of LPS-induced PTB following prophylactic GR1SN treatment

Study Pre-treatment LPS Difference in rates of PTB (%)

Significance (P<0.05)

(Yang et al. 2014) GR1SN-MRS (IP) LPS (IU) 43% Yes

Current GR1SN-MRS (IP) LPS (IP) 12% No (P=0.4)

GR1SN-PBS (IP) LPS (IP) n/a No (P=0.58)

The mode of LPS administration is an obvious variable in in vivo experiments. The

previous study by Yang et al. focused on the ability of GR1SN-MRS administered

intraperitoneally to attenuate the effects of local, IU LPS infusion, which was performed by

mini-laparotomy procedures. In the present study, I tried to extend these effects to observe

GR1SN-mediated attenuation of immune responses following systemic LPS administration.

However, there are two influential variables to be considered following surgery that are absent in

the current study; wound healing and surgical stress response.

Wound healing is a long-term process starting with blood coagulation, inflammation, re-

epithelialization, and ending with matrix and tissue remodelling. Inflammation is an early wound

healing response element, during which TGF-b functions as a pro-inflammatory mediator to

promote leukocyte recruitment and activation by inducing secretion of IL-8, MCP-1, TNF-a, IL-

1b, and platelet derived growth factor (PDGF) in an autocrine manner. This pro-inflammatory

64

response and leukocyte recruitment peaks 24 to 48 hours post-wound, (Leibovich and Ross,

1975; Koh and DiPietro, 2011) after which TGF-b regains its anti-inflammatory characteristic

and promotes production of anti-inflammatory factors such as IL-4. (Wahl, 1994)

Conversely, surgical stress is the systemic response that causes immunosuppression and

additional perturbations in genomic, metabolic, and hormonal regulation following surgical

injury. Glucocorticoid, an upregulated hormone in response to surgical stress, is a known

mediator of anti-inflammatory signaling, which can repress wound healing and expression of

pro-inflammatory factors. Furthermore, glucocorticoid activity has been demonstrated in

relevance TLR regulation to synergistically induce TLR2 expression with TNF-a and IL-1b,

(Hermoso et al., 2004; Sakai et al., 2004; Shibata et al., 2009) suggesting a potential role in

TLR2-mediated endotoxin tolerance. Interestingly, a recent study elucidated the role of

glucocorticoids in endotoxin tolerance in its ability to upregulate miRNA-511-5p expression in

human monocytes which directly reduces TLR4 signaling activity, and downstream production

of pro-inflammatory cytokines (Curtale et al., 2017). Together, the systemic responses induced

by wound healing and surgical stress add an additional layer of anti-inflammatory signaling

through TGF-b and glucocorticoids, which may have contributed to immune suppression in

pertinence to the delay of PTB induced by IU-LPS.

In addition to the absence of such events that promote anti-inflammatory signaling

following surgery, we can further speculate that the effect of GR1SN in the amount administered

is insufficient to suppress the effects of IP-LPS. IP-LPS activates systemic inflammation, which

induces a much stronger inflammatory response than IU-LPS through activation of peripheral

leukocytes and adjacent tissues in addition to local uterine tissue and its resident leukocytes.

Previous studies have demonstrated that murine macrophages treated with GR1SN demonstrated

65

preferential production of G-CSF in extremely high concentrations (Meshkibaf et al., 2015),

which promotes suppression of LPS-induced TNF-a expression in adjacent cells in a paracrine

manner (Kim et al., 2006). Therefore, it can be suggested that GR1SN in the amount

administered cannot prime all macrophages that will later be stimulated by LPS to promote G-

CSF production.

4.2.2 Tissue specificity of Lactobacilli effect

In section 3.6, I demonstrated that GR1SN contains unique factors not present in SN of

other commensal lactobacilli (L. rhamnosus GG, L. lactis, L. casei, L. reuteri RC-14) that

suppresses LPS-induced cytokine secretions by myometrial cells in vitro, thus indicating tissue-

specific effects of lactobacilli SN. In vivo, IP injection of GR1SN affects many more cell types

than solely myometrial cells. Previous in vitro studies have elucidated the anti-inflammatory

effect of GR1SN in human trophoblasts (Yeganegi et al., 2010), decidual cells (Li et al., 2014),

amniotic cells (Koscik et al., 2017), and murine macrophages (Meshkibaf et al., 2015). However,

the effect of L. rhamnosus GR-1 in cytokine regulation in other tissue types such as the intestine

are limited. One study reported resolved diarrhea, flatulence, and nausea in 12 HIV/AIDS

patients who took oral supplements of L. rhamnosus GR-1 and L. reuteri RC-14 for 15 days,

(Anukam et al., 2008) while another study examining the effects of oral L. rhamnosus GR-1 and

L. reuteri RC-14 supplements for 14 days in healthy women found no changes in serum IgG,

IgA, or IgM, slight increases in IL-6 and IFN-g that remained within normal ranges, and

undetectable levels of IL-2 and IL-4 (Gardiner et al., 2002). Therefore, it can be speculated that

GR1SN does not elicit anti-inflammatory effects or induce endotoxin tolerance in all tissues,

resulting in persistent high cytokine secretions following IP-LPS administration and eventual

PTB.

66

4.2.3 Compositional differences between GR1SN-MRS and GR1SN-PBS

Although both GR1SN-MRS and GR1SN-PBS did not delay PTB induced by systemic

LPS, differences in GR1SN composition should be considered for future experiments. In the

current study, BCA assays of MRS broth and GR1SN-MRS demonstrated 2000 and 4000µg/mL

of protein respectively, allotting for approximately 2000µg/mL of protein secreted from L.

rhamnosus GR-1. Conversely, only 22µg/mL of protein were quantified in GR1SN-PBS, roughly

100 times less protein than GR1SN-MRS. Reviewing the definition of probiotics, “live

microorganisms which when administered in adequate amounts, confer a health benefit on the

host” (FAO and WHO, 2001), I speculate that there was not sufficient quantity of active

components in GR1SN-PBS required to elicit benefits to pregnant mouse. GR1SN-MRS was

collected after a 12-hour incubation period, which was required to culture bacteria from lag to

exponential phase while GR1SN-PBS was collected after only 2 hours of incubation to maintain

the bacterial growth in exponential phase. The difference in incubation times may likely

contribute to the difference in protein quantity.

Additionally, the change in incubation medium from MRS to PBS may have also resulted

in altered protein content. MRS broth contains various factors and nutrients critical for the

growth of lactobacilli strains. It is conceivable that in the absence of these growth factors, GR-1

metabolites change, and that GR1SN-PBS contain different metabolic components than those

found in GR1SN-MRS. More importantly, growth in PBS starves lactobacilli which could lead

to a selective culture whereby only bacteria with mutations to survive nutrient-depleted

environments proliferate, similar to how bacteria grow antibiotic resistance. (Watson et al., 1998;

Nguyen et al., 2011) Starvation of Lactobacillus casei modeled by 0% lactose in growth medium

was shown to suppress catabolic pathways of lactose and galactose, as well as nucleotide and

protein synthesis. (Al-Naseri et al., 2013) Similarly, a study of Lactobacillus brevis

67

demonstrated alterations in glucose and amino acid catabolizing pathways, inducing stress-

response mechanisms. (Butorac et al., 2013) Interestingly, Lactobacillus brevis starvation also

demonstrated that the pH of cultured medium increased after prolonged bacterial starvation due

to decreased acid production, which may indicate altered effects of bacterial SN. (Butorac et al.,

2013)

Overall, active components in GR1SN that delay the onset of LPS-induced PTB in vivo

and suppress LPS-induced inflammatory responses in vitro have yet to be determined. Through

my work, I have suggested a potential mechanism of GR1SN-mediated endotoxin tolerance

dependent on the TLR2 receptor. I have emphasized the specificity of host-microbiome

interactions by demonstrating GR1SN as a unique prophylactic agent capable of suppressing

LPS-induced cytokine secretions in vitro. My in vivo work suggests that prophylactic GR1SN

administration cannot offer protection against systemic inflammation, and that an adequate

amount of active component may be required to elicit beneficial effects.

4.3 Future Directions

My current in vitro work demonstrates LPS-induced cytokine suppression by Pam3CSK,

a synthetic triacylated lipoprotein, that mimics the amino end of bacterial lipoproteins. From this,

I speculate that the active component of GR1SN may be a triacylated lipoprotein. To test this

hypothesis, L. rhamnosus GR-1 could be cultured with globomycin, an inhibitor of the lipidation

process in lipoprotein synthesis. In the event that GR1SN derived from cultures with globomycin

fail to elicit the suppression of LPS-induced cytokines as was observed in my study, this may

confirm the functional capabilities of bacterial lipoproteins.

We demonstrated previously that inflammation is a key driver of uterine contraction. The

involvement of inflammatory pathways in labour involves the production of other labour-

68

associated molecules such as PGs, MMPs, or OXTR, in addition to increased expression of

cytokines. Thus, it may be interesting to observe how these molecules are influenced by GR1SN

pre-treatment. Furthermore, work by Hutchinson et al. demonstrates that there may be an

involvement of an independent Rho/ROCK pathway to directly induce myometrial contractions.

It is interesting to observe if GR1SN can influence this pathway in the myometrium. (Hutchinson

et al., 2014)

Previous work from our lab has demonstrated that static myometrial stretch was a potent

inducer of various inflammatory markers including pro-inflammatory cytokines including IL-6

and IL-8. Thus, it may be interesting to investigate whether the positive effects of GR1SN pre-

treatment remains limited to mechanisms of endotoxin tolerance, or if its effects are more global

in regulating cytokine suppression linked to the onset of labour.

In vivo, several experiments could test this possibility. First, it would be useful to test

whether prophylactic GR1SN-PBS could prevent the onset of local LPS-induced PTL. This

would more closely resemble previous work by Yang et al. that demonstrated prophylactic

GR1SN-MRS as able to prevent intrauterine LPS-induced PTL. Furthermore, I had hypothesized

that the ineffectiveness of GR1SN-PBS in vivo may be attributed to insufficient quantities of

active component of GR1SN-PBS, which contained 100 times less protein than GR1SN-MRS

used by Yang et al. Thus, it will be important to test concentrated forms of GR1SN-PBS which

match the protein concentrations found in GR1SN-MRS.

69

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