Probiotics for bacterial vaginosis in pregnancy Margaret May Baekalia (PGDipSc) A thesis submitted for the degree of Master of Science At the University of Otago Dunedin New Zealand April 29th 2011 Abstract Bacterial vaginosis (BV) is one of the infections that predispose pregnant women to preterm labour and post delivery complications. The use of antibiotics to treat the BV is not effective with high recurrent cases and antibiotic resistance. The alternative approach is to use Lactobacillus strains as potential probiotics for prophylaxis or for treatment of BV which is an option that generated this study. Six Lactobacillus strains were explored in terms of their capacity to inhibit two BV bacterial growths and to modify immunological responses the BV strains induced in the host. The THP-1 cell line and monocyte derived dendritic cells were used in vitro in order to closely resemble the in vivo situation of host. These cultured cells were exposed to BV bacteria alone or in combination with various strains of lactobacilli. Supernatants from these cultures were assayed for proinflammatory and anti-inflammatory cytokine content. Lactobacillus acidophilus was found to have the most potential as a probiotic to inhibit the BV bacteria using the bacterial interaction studies. The cytokines induced by the lactobacilli demonstrated that the different Lactobacillus strains affected the immune responses differently. They were found to induce both the pro-inflammatory and antiinflammatory responses. An interesting trend towards the production of IL-10 was observed following exposure both THP-1 and monocyte-derived dendritic cells to L. gasseri and L. rhamnosus in combination with the BV bacteria. This promising result indicated that further research may lead to the identification of a potential probiotic to protect pregnant women who have BV. I Acknowledgments I would like to gratefully thank my supervisor Dr Heather Brooks and my co-supervisor Professor Margaret Baird for their assistance, guidance and endless patience throughout this project. Further thanks to Claire Fritzpatrick, Michelle Wilson and Sonia for their technical assistance and advice and Brooks’ lab team 2010-2011 for their continuous laboratory support and team work. To my mother and late father, thank you very much for everything. If it was not for you I would not have been able to achieve all and fulfilled one of my dreams. To my son Eugene, thank you for your unconditional love, understanding and support. And also to my sister, brothers and the whole family thank you for your moral support. I would also like to thank Helen Ferguson, Bobby and Lois Kusilifu, Joy Breward and friends for the time and patience to babysit Eugene. And finally to the NZAID scholarship team (Rebecca Guest and Rebecca Burnip) thank you very much for your moral support and guidance. II Table of contents Page Abstract…………………………………………………………………………………i Acknowledgment………………………………………………………………………ii Table of contents……………………………………………………………………....iii List of Table……………………………………………………………………………ix List of abbreviations…………………………………………………………………...x Chapter 1: Introduction……………………………………………………………….1 1.1 General introduction…………………………………………………………..1 1.2 The normal flora of the female genital tract……………………..……………1 1.3 The protective role of the vaginal lactobacilli species……………...………....2 1.3.1 Production of lactic acid and low pH………………………...….……2 1.3.2 Hydrogen peroxide producing………………………………….…..…2 1.3.3 Bacteriocin substances…………………………………………….......3 1.3.4 Biosurfactants……………………..…………………………………..3 1.3.5 Competitive adherence……………………………………………..…3 1.3.6 Coaggregation and autoaggregation……………………………….….3 1.4 The immune response…………………………………………………...…….4 1.4.1 The role of cytokines in inflammation………………………...………4 1.4.2 The immune response in the female reproduction tract…………...…..5 1.5 Bacterial vaginosis………………………………………………………...…..5 1.5.1 Gardnerella vaginalis…………………………………………………….…6 1.5.2 Atopobium vaginae………………………………………………………..…7 1.6 Biofilm formation…………………………………………………...………...8 1.7 Consequences of BV………………………………………………………….8 1.7.1 BV and infection in non pregnant women…………………...…….…8 1.7.2 BV and obstetric complications……………………………...……….9 1.7.3 Infection and inflammation………………………………..………….9 1.8 Treatment for BV…………………………………………...……………….11 1.9 Probiotics…………………………………………………...………………..12 1.10 Hypothesis…………………………………………………………...……..13 1.11 Aims………………………………………………………………………..13 1.12 Objectives…………………………………………………………...……...14 1.13 Outline of the study………………………………………………………...14 III Chapter 2: Bacterial interactions………………………………….………………...15 2A Introduction……………………………………………….….…………......15 2B Materials and Methods……………………………………..……….….…..16 2B.1 Culture media…………………………………………………..……16 2B.2 Bacterial strains………………………………………………..….…16 2B.3 Storage and culture………………………………………………..…16 2B.3.1 A. vaginae # 38953 and G. vaginalis # 810……….……...…16 2B.3.2 Lactobacillus strains……………………………….……...…17 2B.3.3 E. coli ATCC 25922………………………………...……….17 2B.4 Gram staining of bacterial strains………………………………...….17 2B.5 Standard curve………………………………………………….……17 2B.5.1 Turbid metric measurement (Optical density)….………..…..17 2B.5.2 Viable bacterial count…………………….….………………18 2B.5.3 Standard curve……………………………………………….18 2B.6 McFarland Standards……………………………….………………..18 2B.7 Heat inactivation of bacteria…………………………………………18 2B.7.1 Optimising heat and time to inactivate bacterial strains….….19 2B.8 Preparation of Lactobacillus strains extracts by heat inactivation…...19 2B.8.1 Culture and bacterial count………....…………………….….19 2B.8.2 Heat inactivation and centrifugation of lactobacilli extracts……………………………………………………....20 2B.9 Preparation of BV bacterial extracts by heat inactivation……………20 2B.9.1 Determining bacterial number….…………………………….20 2B.9.2 Heat inactivation and centrifugation of BV bacterial extracts………………………………………….……………20 2B.10 Preparation of bacterial supernatant……………………………..….20 2B.11 Inhibition of BV bacteria by Lactobacillus strains………….………21 2B.11.1 Overlay antagonism assay……….………………….……....21 2B.11.2 Disc diffusion technique………….…………………….…..21 2B.11.3 Spot inhibition method……………..………………….……22 2B.12 Co-aggregation assay……………………………….……………....22 2B.13 Determination of hydrogen peroxide production…………….……..23 IV 2C Results……………………………………….………………………………24 2C.1 Gram staining reaction…….…………………………………………24 2C.2 Heat inactivation of bacterial culture…….………………….……….24 2C.3 Inhibition of BV bacteria by Lactobacillus strains…………………..24 2C.3.1 Overlay antagonism assay……………………….……...……24 2C.3.2 Disc diffusion assay…………………………………….……24 2C.3.3 Spot inhibition test……………………….……….….………25 2C.4 Co-aggregation Assay………………………………….….…………25 2C.5 Hydrogen peroxide production………………………....……………25 2D Discussion……………………………………………………………………31 2D.1 Heat inactivation…………………………………….…….…………31 2D.2 Inhibition assay……………………………….…………..……….…31 2D.3 Co-aggregation assay…………………………………….…..………32 2D.4 Hydrogen peroxide producing……………………………….………32 Chapter 3: Immune responses……………………………………………………….34 3A. Introduction………………………………………………………………...34 3A.1 Investigation of cytokine response to Lactobacillus strains and BV bacteria ………………………..……………………………………..34 3A.2 The human monoblastic leukaemic cell line (THP-1)….……………35 3A.3 Monocytic derived dendritic cells (MD-DCs) and Langerhans cells (MD- LCs)...........................................................................................36 3A.4 Anti-inflammatory cytokines………………….…………..…………36 3A.5 Pro-inflammatory cytokines……………….…………………………37 3A.6 Aims……………………………………….……………...………….37 3B Materials and Methods……………………………………………………..38 3B.1 THP-1 cell line…………………………………………….…………38 3B.2 Resuscitation and subculture of THP-1 cells…………………...……38 3B.3 Preparation of PMA………………………………………………….39 3B.4 Preparation of LPS and LTA…………………………………………39 3B.5 Optimising PMA dose to differentiate THP-1 cells from monocytes to macrophages………………………………………………………39 3B.6 Tests to confirm differentiation of THP-1 cells………………….…..40 3B.6.1 Crystal violet staining….………...…………………………..40 3B.6.2 Viable count of THP-1 cells by trypan blue exclusion…...….40 V 3B.7 Optimising THP-1 cell concentration………………………….…….41 3B.8 Optimising dose of LPS and LTA………………….…………….…..41 3B.9 Optimising lactobacilli and BV bacteria concentration on THP-1 cells……………………………………………………….…41 3B.10 Assessment of cytokine response of differentiated THP-1 cells following exposure to bacteria……………………..………..41 3B.11 Culture of human peripheral blood monocytes………………….….42 3B.11.1 Harvesting blood monocytes…………………………….....42 3B.11.2 Generating dendritic cells from CD+14 monocytes………..43 3B.12 MD-DCs challenge with BV bacteria and Lactobacillus strains………………………………………………….………...…43 3B.13 TNF-α cytokine ELISA……….…………………………………….43 3B.14 Bio-Plex assay for cytokines IL-1β, IL-6, IL-8, IL-10 and IL-12……………………………………………………………….44 3B.14.1 Principle of assay……………………….………………….44 3B.14.2 Preparation of reagents for immunoassay……………….…45 3B.14.2.1 Preparation of antibody immobilised beads………..45 3B.14.2.2 Preparation of quality control and wash buffer.........45 3B.14.2.3 Preparation of human cytokine standard…………...45 3B.14.2.4 Bio-Plex immunoassay procedure………………….45 3B.15 Bio-Plex assay for cytokine TGF-β1………………………………..46 3B.15.1 Preparation of reagents for immunoassay……………….….46 3B.15.1.2 Treatment of cell culture supernatants containing serum………………………………………………..46 3B.15.1.3 Bio-plex immunoassay procedure for TGF-β1….....46 3C Results……………………………….………………………………………47 3C.1 Determining the PMA dose to differentiate THP-1 cells from monocytes to macrophage………………..………………….………47 3C.1.1 Crystal violet staining and Absorbance reading…….……..…47 3C.1.2 Viable cell count by trypan blue………………………….….47 3C.1.3 Determining the THP-1 cell concentration……………….….47 3C.1.4 Lactobacilli and BV bacteria concentration on THP-1 cells for the generation of TNF-α….…………………….………..48 3C.2 ELISA assay for TNF-α production by THP-1 cells stimulated by VI Lactobacillus strains and BV bacteria……………………………….54 3C.3 Preliminary experiments to determine TNF-α production from human MD-DCs MD-LCs by ELISA assay………………...……………….54 3C.4 TNF-α production by MD-DCs from 3 different female donors measured by ELISA………………………………………..………..55 3C.5 Bio-Plex immunoassay results…………………………………...…..59 3C.5.1 Anti-inflammatory cytokines……………….……..……...….59 3C.5.1.1 TGF-β1 produced by THP-1……….………..………59 3C.5.1.2 IL-10 produced by THP-1………….………………..59 3C.5.2 Pro-inflammatory cytokines………………….………..……..60 3C.5.2.1 IL-1β produced by THP-1 …………………………..60 3C.5.2.2 IL-6 produced by THP-1………………….…………60 3C.5.2.3 IL-8 produced by THP-1…………………….………60 3C.5.2.4 IL-12 produced by THP-1………………….………..60 3C.5.2.5 TNF-α produced by THP-1…………….…….…..….61 3C.6 Preliminary Bio-Plex assay using supernatants from MD-DCs ……..66 3C.6.1 Anti-inflammatory cytokines……………….……….……….66 3C.6.1.1 TGF-β produced by MD-DCs …………………...….66 3C.6.1.2 IL-10 produced by MD-DCs …………………..……66 3C.6.2 Pro-inflammatory cytokines……………………..…...............66 3C.6.2.1 IL-1β produced by MD-DCs……………………..….66 3C.6.2.2 IL-6 produced by MD-DCs……………………….…66 3C.6.2.3 IL-8 produced by MD-DCs………………….………67 3C.6.2.4 IL-12 produced by MD-DCs…………………..…….67 3C.6.2.5 TNF-α produced by MD-DCs…………………….....67 3D Discussion……………………………………………………………………75 3D.1 THP-1 cells, MD-DCs and MD-LCs……………………...…………75 3D.2 Optimisation assay and preliminary testing………………….………75 3D.2.1 Optimising PMA and THP-1 cell concentration…….….……76 3D.2.2 Optimising bacterial load, LPS and LTA to trigger TNF-α response……………………………………………..76 3D.3 TNF-α production by THP-1 cells in response to Lactobacillus strains and BV bacteria measured by ELISA……………………….………77 3D.4 TNF-α production by MD-DCs from 3 female donors in response to VII Lactobacillus strains and BV bacteria measured by ELISA ……..…78 3D.5 Anti-inflammatory cytokines produced by THP-1 cells in response to Lactobacillus strains and BV bacteria measured by Bio-Plex Assay……………………………………………………………...…79 3D.6 Pro-inflammatory cytokines produced by THP-1 cells in response to Lactobacillus strains and BV bacteria measured by Bio-Plex Assay……………………………………………………………...….80 3D.7 Anti-inflammatory cytokines produced by MD-DCs in response to Lactobacillus strains and BV bacteria measured by Bio-Plex Assay………………………………………………………….…..…81 3D.8 Pro-inflammatory cytokines produced by MD-DCs in response to Lactobacillus strains and BV bacteria measured by Bio-Plex Assay………………………………………………………………....82 3D.9 Concluding remarks and Future directions……………………...…...82 References…………………………………………………………………..…………83 Appendices A Standard curves of Lactobacilli and BV bacteria...……………………….…....101 B TMB-plus media………………………………………………………………..105 C ELISA reagents…………………………………………………………………106 D Bioplex assay results……………………………………………………………107 VIII List of Tables Page Chapter 2C Table 1 Overlay antagonism assay 26 Table 2 Disc diffusion assay 27 Table 3 Spot inhibition assay 28 Table 4 Co-aggregation assay 29 Table 5 H2O2 production by Lactobacillus strains 30 IX List of abbreviations APC antigen-presenting cell A. vaginae Atopobium vaginae BD Becton Dickinson BHI Brain heart infusion broth BV Bacterial vaginosis Cfu/ml Colony forming unit/millilitres CO2 Carbon dioxide DC Dendritic cells DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide E .coli Escherichia coli ELISA Enyme-linked immunosorbent assay FCS foetal calf serum g gram GM-CSF Granulocytic macrophage colony- stimulating factor GTI Genital tract infections G. vaginalis Gardnerella vaginalis HBA Human blood agar HIV human immunodeficiency virus h hour H2 O2 hydrogen peroxide IFN-γ interferon-gamma IL-1α interleukin 1-α IL-1β interleukin 1-beta IL-4 interleukin-4 IL-6 interleukin-6 IL-8 interleukin-8 IL-10 interleukin-10 IL-12 interleukin-12 LPS lipopolysaccharide LTA lipoteichoic acid L. acidophilus Lactobacillus acidophilus X L. casei Lactobacillus casei L. crispatus Lactobacillus crispatus L .gasseri Lactobacillus gasseri L. paracasei Lactobacillus paracasei L. reuteri Lactobacillus reuteri L. rhamnosus Lactobacillus rhamnosus MD-DCs monocytic derived dendritic cells MD-LCs monocytic derived Langerhans cells mg milligram mm millimetre ml millilitre MRS de-Man-Rogosa-Sharpe agar NF-κB Nuclear factor-kappa B N. gonorrhoea Neisseria gonorrhoea ng nanogram OD Optical density PAMPs Pathogen-associated molecular patterns PBMCs peripheral blood mononuclear cells PBS phosphate buffered saline pg picogram PID Pelvic Inflammatory diseases PMA phorbol 12-myristate 13-acetate PROM Premature rupture of membranes PSD Peptone starch dextrose Rcf relative centrifugal force Rpm rotations per minute RPMI Roswell Park Memorial Institute-synthetic tissue culture Medium SBA Sheep blood agar STI Sexually transmitted infections TGF-β1 tumour growth factor-beta 1 TH1 T helper 1 cells TH2 T helper 2 cells TLR Toll-like receptors XI TMB 3,3’,5 ,5’ –tetramethylbenzidine dihydrochloride TNF-α tumour necrosis factor-alpha ul microlitre UTI Urinary tract infections VVC Vulvovaginal candidiasis VD3 1, 25 dihydroxyvitamin D3 XII Chapter 1 1. INTRODUCTION 1.1 General Introduction The vaginal microflora was first described as predominantly Gram positive rods in 1892 by Albert Dörderlein. The rods were named as Dörderlein bacilli which were later classified as Lactobacillus species by Thomas (Forsum et al., 2005). It is evident from many studies that Lactobacillus species are the dominant bacteria within the female genital tract microflora. Other anaerobic and aerobic bacterial species are also present within the microflora and are kept in control by lactobacilli. The lactobacilli play an important role in maintaining the acidic environment that protects against pathogenic species (Hillier et al., 1992). The lactobacilli are found to colonize mostly the vaginal epithelium, intestinal tract and the oral cavities of humans and non-human animals (Rodendo-Lopez et al., 1990). Moreover, an increased research interest in women’s health over the past decade has provided more knowledge on the microbial ecology of the female genital tract. It has been postulated that imbalance within the microflora leads to infections such as bacterial vaginosis (BV), urinary tract infections (UTI), genital tract infections (GTI), vulvovaginal candidiasis (VVC) and sexually transmitted infections (STI) which can predispose women to having complications during or after pregnancy (Klebanoff et al., 2004). The use of antibiotics in treating these infections has been shown to disturb the normal microflora resulting in recurrent BV and an increase in antibiotic resistant bacteria (Andrade et al., 2006). An alternative suitable therapy would be to replenish the genital tract with lactobacilli after antibiotic treatment (Mastromarino et al., 2002). 1.2 The normal microflora of the female genital tract The normal microflora of female genital tract is to some extent complex and unique between every woman. However, in healthy premenopausal women, it is dominated by Lactobacillus species, which are facultative anaerobes. The major phylotypes are L. crispatus and L. jensenii, or L. gasseri and L. iners which were detected by molecular experiments (Antonio et al., 1999), (Zhou et al., 2004), (Song et al., 1999), (Vásquez et 1 al., 2002), (Fredricks et al., 2005). Similar studies carried out on African women in Nigeria (Anukam et al., 2006a) and Caucasian women in Seattle, USA (Vallor et al., 2001) have also confirmed the presence of these lactobacilli species. Other Lactobacillus species, such as L. vaginalis and L. gallinarum, are found to also colonize the vagina in some women (Witkin et al., 2007). 1.3 Protective Role of the vaginal lactobacilli species It has been suggested that lactobacilli species play a distinct role in maintaining a healthy vagina. They have numerous antagonist properties to protect the human female vagina from colonisation by pathogens, thus possibly preventing genital infections (Boris and Barbes, 2000). The ability to produce various metabolites which limit the growth of other bacterial species with which they co-habitat is also another distinct feature of lactobacilli (Antonio et al., 1999). Thus, the lactobacilli are able to interfere with the growth of pathogens by several mechanisms such as: inhibition of growth; depletion or reduction of available essential nutrients; adherence to epithelial cell surfaces and exclusion of the pathogens; and also modulating the immune responses of the host (Reid and Burton, 2002). These are discussed in more detail below. 1.3.1 Production of Lactic acid and low pH Most of the predominant Lactobacillus species that maintain a healthy vaginal ecosystem in reproductive women are lactic acid producers (Boris et al., 1998). By utilizing the abundant vaginal glycogen in concert with vaginal oestrogen, lactic acid is produced which in turn maintains the vaginal pH at less than 4.5. This acidic condition limits the survival of other commensal bacteria and also inhibits the growth of pathogenic microorganisms (Antonio et al., 1999), (Hawes et al., 1996). A study by Aroutcheva and colleagues has indicated that lactic acid has an inhibitory effect against Gardnerella vaginalis and other anaerobes (Aroutcheva et al., 2001a). 1.3.2 Hydrogen peroxide producing The majority of lactobacilli have been shown to produce hydrogen peroxide (H2O2). This metabolic compound has been shown to inhibit growth of vaginal microbes either directly or through the peroxidise-mediated system (Eschenbach et al., 1989), (Hillier et al., 1992). Hydrogen-peroxide-producing lactobacilli were found to be toxic for 2 bacterial vaginosis microorganisms such as G. vaginalis, Bacteroides, and Prevotella bivia from various in vitro studies and also Neisseria gonorrhoeae (Klebanoff et al., 1991), (Hillier et al., 1992), (Hawes et al., 1996), (Atassi et al., 2006), (Zheng et al., 1994). It is believed that the loss of H2O2 producing lactobacilli leads to subsequent derangement of the healthy vaginal microflora resulting in BV. 1.3.3 Bacteriocin substances Bacteriocins are any group of proteinaceous based toxins produced by most bacteria that produce antagonistic effects against other bacterial strains (Merk et al., 2005). Bacteriocins produced by lactobacilli have a range of inhibitory activity against other bacteria. Narrow-range lactocins mostly target closely related Lactobacillus species, whilst the broad-range lactocins exhibits inhibition against a wide array of bacteria including Escherichia coli and some of the BV organisms (McGroarty, 1993). 1.3.4 Biosurfactants Biosurfactants are compounds produced and released by some Lactobacillus strains which accumulate at interfaces and help the microorganisms to bind to collagen on epithelial cells. They are found to inhibit adhesion of pathogens involved in urogenital tract infections (Velraeds et al., 1996), (Reid et al., 1999 ). 1.3.5 Competitive adherence Vaginal lactobacilli have also demonstrated the capability to adhere to vaginal epithelia and competitively exclude pathogens to enable barrier protection of the vaginal epithelium (Rodendo-Lopez et al., 1990). A study by Boris and colleagues found that some Lactobacillus strains elicit adherence to vaginal epithelia by bacterial cell’s glycoprotein and carbohydrate moieties. Lactobacilli have a higher affinity for vaginal cell receptors and compete against G. vaginalis and Candida albicans for attachment sites. Therefore, the pathogens were displaced through the receptor binding interference mechanism (Boris et al., 1998). 1.3.6 Coaggregation and autoaggregation Another characteristic of some Lactobacillus strains is their ability to autoaggregrate and coaggregate with other bacterial species through single-cell or multiple-cell binding mechanism (Boris et al., 1997), (Kmet and Lucchini, 1997). Reid et al demonstrated 3 that lactobacilli coaggregates are adherent to the vaginal epithelial cells (Reid et al., 1990a). Furthermore, Mastromarino and colleagues have shown coaggregation of three Lactobacillus strains with G. vaginalis has inhibitory activity against this pathogen (Mastromarino et al., 2002). 1.4 The immune response The main primary function of the host immune system is to protect against infection. The host immune system reacts primarily with a non-specific response known as inflammation to the infection or injury followed by migration of phagocytic immune cells to destroy the infective agent in the inflamed tissues. Macrophages as antigen presenting cells (APCs) are stimulated to mature and release a network of proinflammatory cytokines after exposure to pathogen by special cellular recognition (Cavallion, 1994), (Gordon and Taylor, 2005). The inducement of pro-inflammatory cytokines like interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) are usual after the initial exposure to bacterial products (Gabay, 2006), (Gordon and Taylor, 2005). The adaptive immune response involves activation of Tcells by antigenic peptide displayed on the surface of APCs followed by subsequent stimulation of antibody production by B cells. The CD4+ T helper cells release cytokines that drives an either a predominantly TH1 response (the cell-mediated immunity) or a TH2 response towards antibody synthesis. The activation of CD8+ cytotoxic T cells, macrophages and natural killer cells depend on the release of TNF-α and interferon γ (IFN-γ) (Hume, 2008). 1.4.1 The role of cytokines in inflammation Cytokines are chemical signals which have an essential role in regulating the inflammatory responses to invading pathogens. These are pleiotropic glycoprotein (826 kD) molecules produced by a range of cells including monocytes, macrophages, dendritic cells (DC), lymphocytes, endothelial cells and fibroblasts. They function in a network of cascade to highly regulate the production of other cytokines. Pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α are known important early mediators of inflammation and infection. They induce production of reactive and cytotoxic species of oxygen and nitrogen involved in bactericidal function. The other 4 two cytokines includes interleukin-8 (IL-8) mainly secreted by macrophages with a role as chemo-attractant of phagocytes to the site of infection and interleukin-12 (IL-12) also produced by both macrophages and DC in response to foreign particles. On the other hand, the production of anti-inflammatory cytokines like tumour growth factorbeta 1 (TGF-β1) and interleukin-10 (IL-10) are involved in inhibition of the proinflammatory cytokines (Boyle, 2005). 1.4.2 The immune response in the female reproductive tract The vaginal epithelial cells are the first contact point with the local microbial flora and forms the integral part to innate and inflammation. The cells not only give the physical barrier to infective microbes but also recognise microbial molecules through Toll-like receptors (TLR) which induce production of pro-inflammatory cytokines like interleukin-1α (IL-1α), IL-8 and TNF-α and β-defensin, an antimicrobial peptide (Fichorova and Anderson, 1999), (Pivarcsi et al., 2005). Moreover, the female reproductive tract immune system has macrophages and dendritic cells as the major antigenic presenting cells (APCs) within the genital mucosal (Laskarin et al., 2007). Under a steady state, the DCs reside within the epithelium as Langerhan’s cells and in the submucosa as submucosal DCs. During an infection or inflammation, the antigens are attached to the DCs and transported to secondary lymphoid tissues to initiate the adaptive immune response. This is followed by release of cytokines by the APCs to peripheral blood to recruit monocytic-derived DCs and plasmacytoid DCs to the vaginal mucosa (Zhao et al., 2003), (Ilijima et al., 2007).The DCs are the most potent APCs and are involved in both the innate and adaptive immune responses. They have the ability to activate naive T-cell responses and drive toward either TH1 or TH2 immune responses (Banchereau et al., 2000). 1.5 Bacterial vaginosis BV was first reported by Duke and Gardner as a clinical condition affecting women of child-bearing age known as Gardnerella vaginitis and was thought to be caused by a single micro-organism. However, in 1983 BV was defined as a synergistic, polymicrobial syndrome due to a complex alteration in the vaginal normal microflora resulting in an increased quantity of anaerobic bacteria (Spiegel, 1991). 5 The shift in the normal microflora is associated with reduction or loss of hydrogenperoxide producing Lactobacillus strains and an increase in the vaginal pH from 3.5-4.5 to 7.0. This is more favourable to overgrowth of the other commensal anaerobes (Hillier et al., 1992). The more alkaline environment favours production of proteolytic carboxylase enzymes by the anaerobes with squamous cell exfoliation and malodorous vaginal discharge with little or no inflammation (Hill, 1993), (Colli et al., 1996), (Sobel, 2000). The most common vaginal anaerobes known to associate with BV include G. vaginalis, Bacteroides, Prevotella species, Mobiluncus species, Mycoplasma hominis, Porphyromonas and Fusobacterium species (Hill, 1993),(Sobel, 2000). Most recently Atopobium vaginae has been found within the microbiota of women with BV using molecular methods and is present in 50 % of BV cases (Fredricks et al., 2005), (Oakley et al., 2008). BV accounts for 40-50% cases of vaginal discharges amongst women of reproductive age. Women with symptomatic BV often present with offensive, fishy-smelling discharge. However, 50 % of cases appear to be asymptomatic (Wang, 2000). The worldwide prevalence of BV ranges between 11-48% in child-bearing age with variation in prevalence rates between populations studied (Tolosa et al., 2006). The prevalence of BV in women attending STI clinics was estimated to be 20-40% and1520% of pregnant women (Sobel, 2000), (Klebanoff et al., 2004). Studies have also revealed that BV has associated risk factors such as black ethnicity/race, cigarette smoking, douching practices, use of intrauterine contraceptive devices and sexual practices including oral sex, number of sexual partners and lesbian sexual activities (O'Brien, 2005). 1.5.1 Gardnerella vaginalis G. vaginalis cells are fastidious, non-motile, beta-haemolytic, noncapsulated gram negative to gram-variable small pleomorphic rods. This organism was first named Haemophilus vaginalis by Gardner and Duke because it was isolated from vaginal samples cultured on blood agar. It was later referred to as Corynebacterium vaginale by Zinnemann and Turner because it does not require X and F factors for growth like the Haemophilus species and also displays some features resembling Corynebacterium. However it was renamed G. vaginalis after two large taxanomic studies carried out in 6 the early 1980s using molecular methods showed that there was less or no similarity to the known gram positive or gram negative genera (Catlin, 1992). A number of studies have suggested that G. vaginalis has distinct features which may assist in its virulence and production of disease. An electron microscopy study on the cell wall structure indicated G. vaginalis is exopolysaccharide producing. Web-like strands were seen connecting adjacent cells and these were described as fibrillar exopolysaccharide which may play a role in adherence of G. vaginalis to vaginal epithelial cells and as a contributing factor to biofilm formation (Sadhu et al., 1989). Numerous studies by Jarosik and colleagues have demonstrated that G. vaginalis uses human lactoferin binding protein to acquire iron from iron-loaded lactoferin compounds (Jarosik et al., 1998), (Jarosik and Land, 2000). Another distinct feature is also the production of cytolysin (later named as vaginolysin by Gelber and colleagues) which acts as a haemolysin. This protein toxin is selective only for human cells through recognition of the complementary regulatory molecule CD59 (Cauci et al., 1993), (Gelber et al., 2008). 1.5.2 Atopobium vaginae A. vaginae bacteria also associated with BV are non-motile, lactic acid producing and anaerobic Gram positive elliptical cocci or rod-shaped organisms (single, pairs or short chains). This species was first isolated from a healthy vaginal microflora from a female in Göteborg, Sweden by a conventional culture based technique and later identified by molecular analysis using comparative 16S rRNA gene sequencing (Jovita et al., 1999). A. vaginae was found to be highly resistant to metronidazole, the therapeutic antibiotic commonly used to treat BV (Menard et al., 2008), (Ferris et al., 2004). In a cross sectional study of BV, 40% of the women had A. vaginae and half of the cases (50%) had co-colonisation with G. vaginalis (Burton et al., 2005). This co-existence suggests a synergestic relationship between A.vaginae and G. vaginalis. This phenomenon was extensively studied by Swidsinski, which he believed was due to the biofilm formating capability of the G. vaginae bacteria (Swidsinski et al., 2005). 7 1.6 Biofilm formation Another significant feature of BV is the formation of biofilm on the surface of the vaginal epithelium. Biofilms are communities of microorganisms which secrete extracellular polymeric substances made of polysaccharides, proteins and/or nucleic acids in which the bacteria co-exist symbiotically (Lindsay and von Holy, 2006). About 95% of the microbiota composition in women with BV consisted of G. vaginalis. Furthermore, composition of the biofilms included A. vaginae and also a diversity of other vaginal commensals. On the other hand, healthy women without BV had Lactobacillus populations associated with vaginal epithelia and no biofilms were observed (Swidsinski et al., 2005). The adherence of G. vaginalis to the exfoliating epithelial cells creating “clue cells” is indicative of biofilm formation and at the same time is taken as microscopic diagnosis of the clinical disorder (Gutman et al., 2005). The physical structure of biofilms increases bacterial resistance to antimicrobials, pH extremes and host immune defences and is an advantage to the resident microbes. As such, the survival of BV associated anaerobes in biofilms within the vagina is an important component of the pathogenesis of BV (Patterson et al., 2007). 1.7 Consequences of BV Although BV is not life threatening, it has been associated with numerous reproductive health complications of women, some of which have potentially serious outcomes. 1.7.1 BV and infection in non pregnant women In non pregnant women BV has been associated with pelvic inflammatory diseases (PID) caused by N. gonorrhoeae and Chlamydia trachomatis including endometritis and salpingitis (Hillier et al., 1996), (Wiesenfeld et al., 2002), (Ness et al., 2005). It has also been found from a number of studies that BV increases the risk of acquiring human immunodeficiency virus (HIV) infection and other sexually transmitted infections such as genital herpes (Sewankambo et al., 1997), (Jamieson et al., 2001), (Cherpes et al., 2003). It has been postulated that due to the lack of H2O2 producing lactobacilli, there is loss of viricidal action upon HIV. Furthermore, the subsequent increase of pH also triggered loss of acidic pH-mediated inhibition of CD4 lymphocytes enabling survival and growth of the virus (Taha et al., 1998). 8 1.7.2 BV and obstetric complications It has been revealed that BV is present in 15-20% of pregnant women who have been screened for BV during pregnancy (Sobel, 2000). BV infection has also been associated with a range of gynaecological and obstetric complications with adverse outcomes in pregnant women from studies around the world. It has found to link with increased risk of serious obstetrical complications including premature rupture of membranes (PROM), spontaneous abortion, preterm birth/delivery and postpartum endometritis, amniotic fluid infection and post-ceasarean wound infections (Hillier et al., 1996), (Hauth et al., 1995), (Jacobsson et al., 2002). Also, second trimester miscarriages due to chorioamnionitis and intra-uterine infections with BV organisms have been linked to low birth weights and high infant mortality rate (McGregor et al., 1995), (Leitich et al., 2003). Furthermore, untreated or unknown status of BV has been shown to increase the risk of post gynaecological surgery infections. Such complications as cuff cellulitis/wound infection following vaginal hysterectomies, post abortion infections and post-caesarean endometritis were reported to be caused by presence of BV organisms (Donders et al., 2000a), (Donders et al., 2000b). 1.7.3 Infection and inflammation It was postulated that intrauterine infection and inflammation associated with BV contributed to precipitating preterm labour by further increasing the production of proinflammatory cytokines; IL-1, IL-6 IL-8 and TNF-alpha in the foetal membranes. The accumulation of proinflammatory mediators increases prostaglandins and proteases, mucinases and sialidases in the chorioamnion, cervix and deciduas. The influx of proteases causes degradation of foetal membranes and cervix extracellular matrix has been associated which leads to preterm labour and premature rapture of membranes (Platz-Christensen et al., 1992), (McGregor et al., 1994). A study being carried out on vaginal fluid and cervical mucus of pregnant women with BV reported high levels of endotoxin and IL-1 (Platz-Christensen et al., 1993). A further study on cervical mucus from pregnant women also indicated presence of endotoxin that induces production of IL-1β, IL-6, TNF-α and fetal fibrinogen from monocytic cell assays (Mattsby-Baltzer et al., 1998). 9 Endotoxin is a toxic heat-stable lipopolysaccharide (LPS) complex present in the outer membrane of gram-negative bacteria that is released from the cell upon lysis. Endotoxin itself is a strong inflammatogenic factor that triggers macrophages to produce prostaglandins and cytokines like TNF-α, IL-1α, IL-1β, IL-6 and IL-8.This supports the notion that gram negative bacteria such as (Prevotella species and Porphyromonas species) contribute to preterm births because they are endotoxin producers. Lipotechoic acid (LTA) is present in Gram positive bacteria and has many of the same properties as endotoxin. However, its presence in BV bacteria has not been established. Furthermore, BV bacteria may also directly induce an inflammatory response through their ability to produce collagenases, elastases and other proteolytic enzymes. These enzymes produced by the BV bacteria can synergistically act to degrade the foetal membranes causing preterm labour (McGregor and French, 1997). The pathogenic microorganism may reach the foetal membranes and amniotic cavity to produce adverse gestation outcomes through a number of routes. These include ascension of bacteria through the vagina and cervix, migration from the peritoneal cavity, dissemination from distant sites by blood through the placenta and during an invasive surgical procedure (Romero et al., 2006). However, uterine infection associated with BV is commonly through the cervix due to the mucin degrading capabilities of the BV bacteria (Denney and Culhane, 2009). In order to prevent rejection of the foetus during pregnancy, there is regulation of the immune responses, including changes in cytokine production. During a normal pregnancy, TH1/TH2 activity is shifted towards TH2. During inflammation and infection there is a shift towards Th1 predominance initiating an inflammatory cytokine cascade resulting in spontaneous abortion, preterm delivery and/or preeclampsia (Challis et al., 2009). It is hypothesized that a similar process, involving ‘sterile inflammation’, induces term labour. Inflammation is not localized and extends to the peripheral blood cells. 10 Until recently, BV was believed to be a non-inflammatory condition so that the underlying reason for the association between BV and PTL remained obscure. However, it has been shown that BV causes cervicitis (Marazzo et al., 2006). Thus, BV is considered to be a precursor of chorioamnionitis, a known risk factor for PTL (Sakai et al., 2004). The most probable sequence of events is as follows. Loss of protective lactobacilli in the vagina results in an increase in pH making conditions favourable for overgrowth of BV bacteria. Bacterial fragments activate Toll-like receptors on macrophages, dendritic cells and fibroblasts, which ultimately lead to changes in gene transcription through the action of NF-kB (Zariffard et al., 2005). Major targets of NFkB are the genes that encode pro-inflammatory cytokines (Sakai et al., 2004). The endpoint is prostaglandin and matrix metalloprotease production which cause tissue remodelling and induces premature labour (Challis et al., 2009), (Diaz-Cueto et al., 2006). 1.8 Treatment for BV As BV is often asymptomatic in women of reproductive age, antibiotic treatment is not always recommended because the balance of the vaginal bacteria may correct itself over time. However, non- pregnant women who experienced vaginal discharge associated with BV are treated either with metronidazole (500 g orally twice daily for 7 days), clindamycin vaginal cream (2%, once daily for 7 days) or metronidazole vaginal gel (0.75%, twice daily for 5 days). Metronidazole (250 mg, 3 times a day) orally is recommended for pregnant women (Centers for Disease Control and Prevention et al., 2006). The cure rate of BV with the recommended antibiotic therapy was found to be significantly poor (50-80%) with a high number of recurrent cases (Joesoef et al., 1999), (Bradshaw et al., 2006). Cases of recurrent BV in a large proportion of women are thought to be mainly due to recalcitrant biofilm formation which is difficult to eradicate with antibiotics (Lewis, 2001), (Swidsinski et al., 2005). With the recurrence of BV despite vigorous antibiotic treatment, this situation brings out the question as to what are the alternate options for treating and curing BV. Apparently a lot of emphasis and consideration has now been focused on lactobacilli 11 due to its inhibitory and protective ability. An alternative approach would be to colonise and replenish women with probiotic lactobacilli after antibiotic treatment. 1.9 Probiotics Probiotics are defined as “live microorganisms which when administered in adequate amounts confer health benefits on the host” (FAO/WHO, 2001). Probiotics have been effectively used for treatment or preventing a number of diseases affecting humans. Some examples of the microbes that are used as probiotics in humans include Saccharomyces boulardii to treat Crohn’s disease and Bacillus subtilis 3 to inhibit Helicobacter pylori. Also included are the commonly used bifidobacteria and lactic acid producing bacteria belonging to the genera Lactobacillus, Lactococcus and Streptococcus (MacFarlane and Cummings, 2002). A variety of probiotics are in popular use and available over the counter as dairy products or food supplements. However, most of these products have no quantified concentration of microorganisms, lack species descriptions and contain non-listed or more than one species (Bolton et al., 2008). Furthermore, the possibility of dairy products contaminated with unwanted bacterial species such as Enterococcus faecium, Clostridium sporogenes, Streptococcus mitis and Pseudomonas species would be problematic (Hughes and Hillier, 1990). A number of criteria are required for an organism to become a suitable probiotic which includes: being non-pathogenic; able to show beneficial effects on the host; isolated from the same species as its intended host; able to survive the gastrointestinal tract (oral formulations); and be able to survive prolonged storage conditions (McNaught and MacFie, 2001). Furthermore, for the probiotics to be protective, a number of essential traits are deemed necessary such as: they should be able to adhere, displace adhesion of pathogen and inhibit their growth; compete for nutrients; produce antimicrobial substances; and modulate host immune responses to reduce risk of infection (MacFarlane and Cummings, 2002). 12 A number of studies have tested the capacity of lactobacilli probiotics to effectively colonise the vagina preventing recurrence of BV. One study included use of L. rhamnosus GR-1 and L. reuteri RC-14 oral formulations administered to female with BV after treating with metronidazole. The results obtained indicated efficacy of combined antibiotic and probiotic treatment in the eradication of BV in black African women (Anukam et al., 2006b). In the present study, different strains of Lactobacillus were evaluated for their probiotic potential using in vitro assays. The first set of assay was concerned with interactions between the Lactobacillus strains and the BV bacteria G. vaginalis and A. vaginae. The second set of assays examined the cytokine response of cells of the immune system to the lactobacilli and BV bacteria both singly and in combination. The cells were chosen on their basis of their importance in the inflammatory processes that lead to the onset of preterm labour. The overall aim of the study was to discover if any of the Lactobacillus strains might be potentially useful as probiotics in pregnancy, both to prevent recurrence of BV and to prevent preterm labour. 1.10 Hypothesis It was hypothesised that potential probiotic Lactobacillus strains would differ in their inhibitory effects against BV microorganisms and in their immunomodulatory activity on cells of the immune system. 1.11 Aims 1. To study the inhibitory properties and effects of Lactobacillus strains on two BV bacteria, G. vaginalis and A. vaginae. 2. To study the effect of selected Lactobacillus strains and two BV bacteria (as above) on cytokine production in a monocytic-macrophage cell line (THP-1) and human monocytice derived dendritic cells (MD-DCs) 13 1.12 Objectives a) To test the Lactobacillus strains against BV bacteria, G.vaginalis and A.vaginae for potential inhibitory action by a number of bacterial interaction assays. b) To test for hydrogen peroxide production in the Lactobacillus strains. c) To investigate the coaggregation capabilities between Lactobacillus strains and BV bacteria. d) To evaluate the optimal heat and time to totally kill Lactobacillus stains and BV bacterial cells. e) To evaluate the optimised PMA dose and time to effectively differentiate THP-1 monocytic cells into macrophages. f) To determine the optimal dose of LPS and lipotechoic acid (LTA) to trigger cytokine production as measured by ELISA assay of TNF-α. g) To investigate the production of proinflammatory cytokines by THP-1 cells, and monocytic-derived DCs cells (MD-DCs) in response to potential probiotic Lactobacillus strains and the BV bacteria G. vaginalis and A. vaginae (alone and in combination). h) To investigate the production of anti-inflammatory cytokines by THP-1cells, and MD-DCs in response to potential probiotic Lactobacillus strains and the BV 1.13 Outline of the study The current study is divided into two parts. Chapter 2 covers studies on interactions between the BV bacteria and the Lactobacillus strains and chapter 3 covers the immune responses of THP-1 cells and MD-DCs to the bacterial strains studied. 14 Chapter 2 2. BACTERIAL INTERACTIONS 2A.1 Introduction The Lactobacillus species are known to have numerous antagonist properties and ability to produce various metabolites to limit the growth of other bacterial species with which they co-habitat or to prevent and inhibit adherence and colonisation of other pathological species (Antonio et al., 1999). The experiments were undertaken to investigate the possible inhibition and interaction between the Lactobacillus strains, G. vaginalis and A. vaginae, the two most common bacteria causing BV. The possible co-aggregation of Lactobacillus strains with BV bacteria and determination of H2O2 production by the Lactobacillus strains were also studied. 15 2B. MATERIAL AND METHODS 2B.1 Culture media The growth media used to cultivate the bacterial strains used in this study were prepared according to the manufacturer’s instructions and autoclaved to sterilise at 121 °C for 15 minutes; de Man-Rogosa-Sharpe (MRS) broth and agar, Peptone Starch Dextrose (PSD) agar (DIFCO, Fort Richard) and 3,3’,5 ,5’ –tetramethylbenzidine dihydrochloride (TMB) plus media (see appendix for ingredients). Human blood agar (HBA) plates and sheep blood agar (SBA) plates were commercially prepared and supplied by Fort Richard Laboratories Ltd, Auckland. 2B.2 Bacterial strains A. vaginae #38953 was obtained from the Göteborg culture collection, Sweden, L. crispatus #20584 from DSMZ, Germany and G. vaginalis #810 from the New Zealand Culture Collection. The six potential probiotic Lactobacillus species were obtained from BLIS Technologies Ltd, Centre of Innovation, University of Otago, Dunedin. The Lactobacillus species were; L. acidophilus, L. paracasei #94.8, L. gasseri #2.1, L. rhamnosus #67B, L. reuteri #61.6 and L. casei GR-1. The L. reuteri strain is known probiotic strain. The remaining lactobacilli strains were chosen because investigations carried out by BLIS Technologies indicated they had some degree of antibacterial activity against BV bacteria. Also included was E. coli ATCC # 25922 which was used in the co-aggregation experiment. 2B.3 Storage and culture 2B.3.1 A. vaginae #38953 and G. vaginalis #810 A. vaginae #38953 and G. vaginalis #810 bacterial strains were resuscitated from stock cultures stored in Brain Heart Infusion broth (BHI) with 20% glycerol at -80 °C. The bacterial strains were thawed and propagated onto HBA plates. A. vaginae #38953 was incubated at 37 °C in an anaerobic atmosphere using the BD GasPak EZ gas generating system (Becton Dickinson, Auckland) for 4 to 7 days whilst G. vaginalis #810 cultures were incubated at 37 °C in a 5% CO2 incubator for 24 to 48 hours (h). 16 2B.3.2 Lactobacilli strains The L. acidophilus strain was received in lypholised form, resuspended in 10 ml of MRS broth and cultured in an anaerobic chamber at 37 °C for 24 to 48 h. The bacterial suspension was then subcultured onto MRS agar and propagated in the same environment as described above. The other Lactobacilli strains were received as colonies on MRS agar plates that were subcultured onto MRS agar plates and incubated anaerobically as previously described. The L. crispatus DSMZ 20584 isolate was thawed from -80°C, inoculated onto MRS broth, subjected to similar growth conditions as the other Lactobacillus stains and further subcultured onto MRS agar. 2B.3.3 E. coli ATCC 25922 This bacterial strain was obtained as colonies on SBA from Dr H. Brooks culture collection (Dept. Microbiology & Immunology, University of Otago), subcultured onto SBA and cultivated at 37 °C, aerobically for 24 h. 2B.4 Gram staining of bacterial strains All the Lactobacillus strains and BV bacteria were Gram stained and examined under high magnification (x100 lens) by light microscopy to confirm the identity of the bacterial growth. 2B.5 Standard Curve In order to standardise the inocula for the bacterial interaction experiments, as standard curve was established. Two commonly used laboratory methods were carried out to produce a standard curve from the optical density versus bacterial number. 2B.5.1 Turbid metric measurement (Optical density) Colonies from each of the Lactobacillus strains were suspended into 10 ml of MRS broth and cultured at anaerobic atmosphere at 37 °C for 48h. The broth cultures were diluted 2-fold in MRS broth and 0.1 ml of each dilution dispensed into a 96 flat-bottom well plate (BD) in triplicate. MRS broth was used as the blank and optical density (OD) was read by absorbance at 595nm on Infinite® M200 plate reader (Tecan, Trading AG, Switzerland). 17 A. vaginae #38953 colonies from a 72 h culture and G. vaginalis #810 colonies from a 48 h plate culture were suspended separately in 10 ml sterile PBS and subjected to the same procedure for Lactobacillus strains as above. Sterile PBS was used as the blank. 2B.5.2 Viable bacterial count For each of the lactobacilli cultures, 10-fold serial dilution was done in MRS broth and 0.1 ml bacteria from each 10-2 to 10-4 dilution were plated onto MRS agar in triplicate, incubated as described above and the number of colonies counted after 48 h. An average of triplicate cultures from the dilution plates showing between 30-300 bacterial colonies was recorded. The BV bacteria, A. vaginae #38953 and G. vaginalis #810 were serial diluted in sterile PBS with triplicate samples from each dilution inoculated onto HBA and incubated in their respective culture conditions. The cfu/ml was obtained as the procedure for Lactobacillus strains as above. 2B.5.3 Standard Curve A standard curve was then created for each of the bacterial strains by plotting the absorbance obtained from each dilution against the cfu/ml obtained from the viable bacterial count using the Microsoft 2007 excel software program. 2B.6 McFarland Standards For the purpose of standardisation of numbers of bacteria (inocula) for heat inactivation experiment the colonies from the bacterial strains were suspended in sterile PBS. The turbidity was then adjusted to the McFarland standards 0.5, 1.0, 2.0 and 3.0, which corresponded approximately to 1.5-9.0 x108 cfu/ml. 2B.7 Heat inactivation of bacteria The determine the optimal temperature and time taken to completely inactivate the bacterial strains, 48 h lactobacilli cultures in MRS broth and BV bacteria suspensions in PBS were exposed to different combinations of heat and time duration in a water bath followed by plating of inocula onto agar plates to check for effective heat inactivation. 18 Sashihara and colleagues have indicated from their study that 75 °C for 60 minutes had inactivated L. gasseri and L. crispatus. Peng & Hsu has demonstrated that between 5690°C was sufficient to heat kill L. paracasei while Gill & Rutherford showed that much higher heat of 100 °C with shorter time duration of 15 minutes (Gill and Rutherfurd, 2001; Peng and Hsu, 2005; Sashihara et al., 2006). From the findings, three sets of heat and time were tested to determine the least heat and times taken to heat inactivate the bacteria. 2B.7.1 Optimising heat and time to inactivate bacterial strains The Lactobacillus strains were each inoculated into 10 mL of MRS broth and incubated as previously described for 48 h. The bacterial broth cultures were examined for turbidity which is indicative of bacterial growth and then exposed to different sets of temperatures and times; 65 °C for 30 minutes and 70 °C for 30 minutes and 75 °C for 60 minutes in a water bath. Since A. vaginae #38953and G. vaginalis #810 are difficult to propagate in broth media, colonies from these bacteria were resuspended to an optical density corresponding to cfu/ml in 10 ml of 1% PBS. These suspensions were then exposed to different temperatures and times: 65 °C for 30 minutes, 70 °C for 30 minutes and 75 °C for 30 minutes in a water bath. The broths were left at room temperature to cool and inoculated onto the appropriate agar plates and incubated as previously described. Examination of the G. vaginalis #810 and the lactobacilli plates for growth was carried out after 48 h whilst A. vaginae #38953 cultures were checked after 72 h. Each broth was cultured on triplicate plates and the experiment was repeated three times. 2B.8 Preparation of Lactobacillus strains extracts by heat inactivation 2B.8.1 Culture and bacterial count The Lactobacillus strains were inoculated separately into 20 ml Universals of MRS broth and incubated for 48 h at 37 °C anaerobically. For each of the lactobacilli, 100 ul were added into a 96 flat bottom well plate (BD) in triplicates and absorbance reading 19 at OD 595 was carried out. Bacterial cells were adjusted to 1x1010 cfu/ml from results obtained from the standard curve (see appendix 1). 2B.8.2 Heat inactivation and centrifugation of lactobacilli extracts The Universals containing 20 ml of lactobacilli cultures were then placed in a water bath of 75 °C for 60 minutes, cooled at room temperature, mixed on the vortex mixer and transferred into a 50 ml BD plastic Falcon tube. The bacterial cultures were then centrifuged at 3,000 rpm for 10 minutes and kept at 4 °C for the cytokine assay after removal of supernatants. 2B.9 Preparation of BV bacterial extracts by heat inactivation 2B.9.1 Determining bacterial number A. vaginae #38953 and G. vaginalis #810 bacterial colonies from HBA were suspended in separate 20 ml sterile PBS vials and adjusted to 1x109 cfu/ml and 1x1010 cfu/ml respectively. This was achieved by reading the absorbance of the bacterial suspensions in a 96 well plate with the OD595 and average of triplicates as that carried out for the lactobacilli. 2B.9.2 Heat inactivation and centrifugation of BV bacterial extracts The Universals containing the BV bacterial suspensions were then placed in the water bath set at 75 °C for 30 minutes. Prior to centrifugation, the bacterial cultures were cooled to room temperature, mixed thoroughly on the vortex mixer, transferred into 50 ml BD plastic Falcon tubes and centrifuged at 1026 relative centrifugal force (rcf) for 10 minutes. The supernatants were removed and the heat killed bacteria kept at 4 °C for the cytokine experiment. 2B.10 Preparation of bacterial supernatant To enable investigation of lactobacilli inhibition on BV bacteria, supernatants from Lactobacillus strains were needed. Each of the Lactobacillus strains were cultivated in a 20 ml MRS broth and incubated as previously described for 48 h. Bacteria count was then obtained from absorbance reading at OD595 and adjusted to 1x109 cfu/ml. The bacterial suspensions were then mixed thoroughly and transferred into 50 ml plastic Falcon tubes (BD) and centrifuged at 1026 rcf for 15 minutes. The supernatants were 20 then collected into sterile 1.5 ml polypropylene Eppendorf tubes and stored at -20 °C for the inhibition experiments. 2B.11 Inhibition of BV bacteria by Lactobacillus strains The inhibitory effects of lactobacillus strains on the BV bacteria were determined using three methods as outlined below. These three methods represent different techniques for detecting extracellular molecules which have an inhibitory effect on the growth of other strains of bacteria. 2B.11.1 Overlay antagonism assay The deferred antagonism assays were prepared according to McLean et al, (McLean and McGroarty, 1996) and were performed to investigate the capability of lactobacilli to inhibit G. vaginalis #810. The method requires cultivation of bacteria to be tested in a broth medium. G. vaginalis #810 was the only BV bacteria tested against the Lactobacillus strains using this method due to the fact that no liquid growth medium was available to support the fastidious growth requirements of A. vaginae The bacterial load of 1x109cfu/ml was determined from reading the bacterial suspension against OD 595nm and estimated from the standard curve created for each of the bacteria (see appendix 1). A 1 ml of 1x109 cfu/ml of 48 hr culture of Lactobacillus from each strain (as determined by optical density measurement) was added to molten cooled MRS agar, poured into a Petri dish and allowed to solidify. An overlay of 10 ml molten PDS agar was poured and allowed to solidify followed by addition of 50 ul of G. vaginalis #810 (1x109 cfu/ml) which was spread on the solidified agar and incubated as previously described for 48 h. Positive and negative control were penicillin 100U/ml and 100ug/ml of streptomycin (1:10 dilution) and sterile PBS respectively. Assessment of inhibition was based on the degree of absence of bacterial growth since this is a qualitative assay. 2B.11.2 Disc diffusion technique After the overlay antagonism assay, the lactobacilli were further investigated for their inhibitory activity against G. vaginalis #810 by the disc diffusion technique. Sterile filter papers discs of 6 millimetres (mm) diameter were impregnated with 25 ul supernatants obtained from each of the Lactobacillus strains as previously described. 21 The filter papers were placed on the PSD agar seeded with pure cultures of G. vaginalis #810 at 9x108 cfu/ ml (MacFarland standard 3.0) and incubated at 37 °C + 5% CO2 incubator for 24 - 48 h. The positive control, penicillin 100 U/ml + 100 ug/ml of streptomycin (Gibco/BRL Life Technologies) was diluted to 1:10 and 1:100 strengths and sterile PBS as negative control were added onto the sterile filter discs and placed onto PSD agar seeded with G. vaginalis #810 and subjected to the same incubation conditions as above and followed by examined for inhibition zones around the impregnated discs. Each test was performed in triplicate and repeated three times with the average recorded. This method was adapted from Fontaine et al, 1996 with some modification (Fontaine et al., 1996). 2B.11.3 Spot inhibition method The spot inhibition test on lactobacilli strains against the BV bacteria was according to the procedure of Ruiz et al 2009 with some modifications (Ruiz et al., 2009). This assay was performed on both G. vaginalis #810 and A. vaginae #38953. A 50 ul filtered supernatant from each of the Lactobacillus strains was spotted on the HBA and airdried for 15 minutes in the 37 °C incubator. The plates were then exposed to UV light in a biosafety class II cabinet (Email airhandling airpureTM hepafilter) for 45 minutes. Colonies of A. vaginae #38953 or G. vaginalis #810 were suspended in sterile 5 ml PBS to McFarland standards of 0.5 (1.5x108 cfu/ml) and seeded onto HBA using sterile cotton swabs. The culture plates were then incubated at their respective growth conditions for 24, 48 and 72 h and finally examined for inhibition zones. Each Lactobacillus strain supernatant was tested in triplicate and the experiment was repeated 3 times with the average recorded, including a positive control of penicillin 100 U/ml and 100 ug/ml of streptomycin diluted to one-tenth strength (Gibco/BRL Life Technologies) and sterile PBS as the negative control. 2B.12 Co-aggregation Assay To measure the capacity for interaction between bacteria, the Lactobacillus strains were tested for their ability to co-aggregate with the two BV bacteria, A. vaginae #38953 and G. vaginalis #810. This assay was performed according to Reid et al 1990 with some modifications (Reid et al., 1990b). The bacterial inocula from 48 h bacterial growth on 22 MRS agar for lactobacilli and HBA for BV bacteria were prepared in sterile PBS to a McFarland standard density of 3.0 (9.0x108cfu/ml). For each of the lactobacilli, 500 ul was combined with 500 ul of either of the BV bacteria in a 24 well tissue culture plate (BD) and gently mixed for 10 seconds by hand followed by incubation at 37 °C on an orbital shaker at 100 rotations per minute (rpm) for 4 h. Auto-aggregation was also assessed for each of the bacterial strains with 500 ul of bacterial suspension mixed with 500 ul of sterile PBS adjusted to the same McFarland standard as above. The positive control used was L. casei GR-1 and E. coli ATCC 25922 which are known to coaggregate (Reid et al., 1990b). The assay was performed in duplicate and the experiment repeated twice. The suspensions were then observed macroscopically and under inversion light microscopy (x16 magnification lens) for aggregation and scored according to the following scale: 0 = no aggregation, 1=small aggregates with visible small clusters of bacteria, 2= aggregates with large numbers of bacteria settling to centre of the well, 3= macroscopically visible clumps of larger group of bacteria that settle to centre of the well, 4= large macroscopically visible clump of bacteria in the centre of the well (Reid et al., 1990b). 2B.13 Determination of H2O2 production In order to identify H2O2 producing lactobacilli, the 6 strains were cultured on TMBPlus agar plates and incubated in an anaerobic incubator at 37°C for 24-72 h (Rabe and Hillier, 2003). The plates were then exposed to ambient air over time in which the colonies were assessed for development of blue pigments. A strong reaction is seen as a colour change within the first 5 minutes, an intermediated reaction is within 5-10 minutes and a weak reaction is 10 to 20 minutes (Rosenstein et al., 1997). This semi-quantitative assay to detect H2O2 lactobacilli producers was modified by Rabe et al 2003 to the previous method described by Eschenbach et al 1989. The ingredients for this medium are described in appendix 2. 23 2C RESULTS 2C.1 Gram staining reaction It is necessary to determine the Gram staining reaction of all bacterial cultures to confirm pure growth. The Lactobacillus strains appear as Gram positive long rods whilst G. vaginalis was stained as Gram variable coccobacilli and A. vaginae as Grampositive to variable elliptical cells in pairs and short chains. 2C.2 Heat inactivation of bacterial culture It was initially necessary to heat inactivate the bacteria strains to release the cell wall products before testing them against the THP-1 cells. From the three sets of time and temperature being tried, the minimum time and heat to totally kill Lactobacillus strains was 70 °C for 60 minutes, whilst for A. vaginae and G. vaginalis it was 70°C for 30 minutes. This was confirmed by no growth of bacterial colonies on the respective agar plates after 48 h for Lactobacillus strains and G. vaginalis and after 72 h for A. vaginae. 2C.3 Inhibition of BV bacteria by lactobacillus strains 2C.3.1 Overlay antagonism assay The results of the overlay antagonism assay (Table 1) indicated that some degree of inhibitory action was produced by all the lactobacilli strains tested. The degree of inhibition varied between the lactobacilli with L. acidophilus presented moderate inhibition against G. vaginalis #810 whilst the other lactobacilli showed mild inhibition activity. The positive control (1:10 diluted strength of penicillin + streptomycin) has markedly inhibited the growth of G.vaginalis #810 whilst the negative control consisted of sterile PBS has no inhibition. 2C.3.2 Disc Diffusion assay Table 2 shows that there was inhibition of G. vaginalis #810 demonstrated by zones of inhibition around the filter paper discs impregnated with the lactobacilli. The highest inhibition activity was displayed by L. acidophilus with 11 mm followed by the other lactobacilli with 10 mm and L. paracasei # 94.8 with the least inhibition of 9 mm. In 24 comparison with the negative control, positive control (antibiotic 1:10 dilution) has 30 mm zones of inhibition. 2C.3.3 Spot inhibition test Most of the lactobacilli show some degree of inhibitory activity against G. vaginalis #810 and A. vaginae #38953 in the spot inhibition assay (Table 3). An inhibition zone of 9 mm diameter was observed for L. acidophilus whilst 4 mm inhibition zone was seen for L. paracasei #94.8 against G. vaginalis #810. There was only partial inhibition observed for all the lactobacilli against A. vaginae #38953. The positive control (penicillin/streptomycin) completely inhibited both of the BV bacteria whilst 1:100 dilution had no inhibitory activity at all. 2C.4 Co-aggregation Assay Results for the coaggregation assay are shown in Table 4.0. There was some degree of coaggregation observed between the lactobacilli and the BV bacteria ranging from a maximum aggregation score of 4 to a least aggregation score of 1. L. acidophilus was observed to be in maximum aggregation with A. vaginae #38953 whilst L. gasseri #2.1 aggregated more with G. vaginalis #810. Autoaggregation was observed for L. acidophilus and L. gasseri #2.1 with the score of 2 which indicated which indicated large numbers of bacteria seen at the centre of the well. L. crispatus DSMZ 20584 and L. reuteri #61.6 has minimum autoaggregation score of 1 with visible small clusters of bacterial aggregates (Table 5.0). 2C.5 H2O2 production To distinguish between H2O2 lactobacilli producers the TMB-plus media was used to grow the bacterial strains. As shown in Table 5.0, L. crispatus DSMZ 20584 was found to be the significant H2O2 producer, a blue colour being observed immediately within 3 minutes after the colonies on TMB-plus was exposed to ambient air. The other two namely L. acidophilus and L. reuteri #61.1 colonies had blue pigments between 5- 10 minutes whilst the rest of the lactobacilli did not produce blue pigment colonies. 25 Table 1 Growth inhibition of G. vaginalis by Lactobacillus strains in the overlay antagonism assay Lactobacillus strains L. acidophilus G. vaginalis #810 ++ L. crispatus DSMZ 20584 + L. reuteri #61.6 + L. gasseri #2.1 + L. rhamnosus #67B + L. paracasei #94.8 + Positive control (1:10 dilution antibiotic) Negative control (PBS) +++ 0 Key: +++ = marked inhibition, ++ = moderate inhibition, + = mild inhibition, 0 = no inhibition 26 Table 2 Growth inhibition of G. vaginalis by Lactobacillus strains using the disc diffusion method Diameter of inhibition zones (mm) Lactobacillus strains G.vaginalis #810 L. acidophilus 11 L. crispatus DSMZ 20584 10 L. reuteri #61.6 10 L. gasseri #2.1 10 L. rhamnosus #67B 10 L. paracasei #94.8 9 Positive control (1:10 dilution) Negative control 30 0 27 Table 3 Growth inhibition of A. vaginae and G. vaginalis by Lactobacillus strains in the spot inhibition assay Diameter of inhibition zones (mm) Lactobacillus strains A. vaginae #38953 G. vaginalis #810 L. acidophilus Partial inhibition 9 L. crispatus DSMZ 20584 Partial inhibition 8 L. reuteri #61.6 Partial inhibition 7 L. gasseri #2.1 Partial inhibition 7 L. rhamnosus #67B Partial inhibition 7 L. paracasei #94.8 Partial inhibition 4 Positive control (1:10 dilution of antibiotic) Complete inhibition Complete inhibition (1:100 dilution of antibiotic) No inhibition No inhibition Negative control (PBS) No inhibition No inhibition Positive control: 100 U/ml of penicillin + 100 ug/ml streptomycin, Negative control: Sterile PBS, Partial inhibition: some bacterial growth observed within the inhibition zone 28 Table 4 Coaggregation between lactobacilli, A. vaginae and G. vaginalis Coaggregation Lactobacillus strains Autoaggregation A. vaginae #38953 G. vaginalis #810 PBS L. acidophilus 4 3 2 L. crispatus DSMZ 20584 1 2 1 L. reuteri #61.6 1 1 1 L. gasseri #2.1 3 4 2 L. rhamnosus #67B 1 1 0 L. paracasei #94.8 1 1 0 PBS 0 0 - + L. casei GR-1) - 3 - A. vaginae #38953 - - 0 G. vaginalis #810 - - 0 Positive control ( E. coli ATCC 25922 Score range scale: 0 = no aggregation, 1= small aggregates with visible small clusters of bacteria, 2= aggregates with large numbers of bacteria settling to centre of the well, 3= macroscopically visible clumps of larger group of bacteria that settle to centre of the well, 4= maximum aggregation (Reid et al., 1990b). 29 Table 5 H2O2 production by Lactobacillus strains Lactobacillus strains H2O2 production L. acidophilus + L. crispatus DSMZ 20584 ++ L. reuteri #61.6 + L. gasseri #2.1 - L. rhamnosus #67B - L. paracasei #94.8 - Key: +, weak producer of H2O2; + +, strong producer of H2O2; −, non producer of H2O2 30 2D DISCUSSION 2D.1 Heat inactivation A number of studies have indicated the optimal conditions to totally inactivate some lactobacilli strains were 75 °C for 60 minutes (Sashihara et al., 2006), 56-90 °C for 30 minutes (Peng and Hsu, 2005) or 100 °C for 15 minutes (Gill and Rutherfurd, 2001). However, in the present study, 75 °C for 60 minutes was adequate to effectively kill the lactobacilli and 75 °C for 30 minutes for the BV bacteria. 2D.2 Inhibition assay The purpose of carrying out the three inhibition assay methods was to investigate the possible inhibitory activity of the potential probiotic Lactobacillus strains against BV bacteria. The overlay antagonism assay performed on the Lactobacillus strains against G. vaginalis has shown some degree of inhibition. The results indicated that L. acidophilus has moderate inhibitory activity for G. vaginalis compared with the other Lactobacillus strains tested, which showed mild inhibition. This was also seen in the disc diffusion assay in which L. acidophilus displayed the largest inhibition zone against G. vaginalis. Furthermore, L. acidophilus also demonstrated a larger zone of inhibition in the spot inhibition assay. The possible reasons for the Lactobacillus strains inhibiting BV bacteria could be due to the production of lactic acid, bacteriocins, lactocins and/or H2O2. However, Due to time constraints, the reasons for the inhibitory activity of the lactobacilli were not investigated in further experiments. Lactic acid is known to be produced by all of the Lactobacillus strains and maintains the low pH of the vaginal microenvironment. Some previous studies have indicated that lactic acid is readily diffusible from bacterial cells upon propagation and it has been shown to inhibit BV bacteria. McLean and McGroaty showed that lactic acid inhibits G. vaginalis NTC 11292 (McLean and McGroarty, 1996). 31 Bacteriocins or lactocins have also been shown to play a role in the inhibition of G. vaginalis in vitro. A study by Aroutcheva had found that 80% of the 22 Lactobacillus strains studied produced bacteriocin and that L. acidophilus 160 inhibited G. vaginalis (Aroutcheva et al., 2001b). Furthermore, Simoes et al demonstrated that through bacteriocin production L. acidophilus strain inhibited 78% of G. vaginalis isolates (Simoes et al., 2001). 2D.3 Coaggregation assay The interaction between bacteria was measured by the potential coaggregating capability of the lactobacilli and BV bacteria. Of the lactobacilli tested in this study, L. acidophilus had maximum aggregation with A. vaginae and formed large visible clumps of aggregates with G. vaginalis whilst L. gasseri showed maximum aggregation with G. vaginalis and large visible clumps with A. vaginae. These 2 lactobacilli have shown potential probiotic functions by coaggregating with BV bacteria in PBS suspension. However, the test could be improved and would better resemble an in vivo environment if the investigation into their coaggregation ability used vaginal epithelial cells in vitro, as discussed below. It has been found from some studies that coaggregation is a mechanism exhibited by lactobacilli to block adherence or displace G. vaginalis from vaginal epithelial cells. According to Boris et al 1998, L. acidophilus, L. gasseri and L. jensenii displayed coaggregation with G. vaginalis on vaginal epithelial cells and reduced adherence as well as displacing previously adherent G. vaginalis (Boris et al., 1998). Another study indicated L. gasseri 335 coaggregated with G. vaginalis in vitro and also displaced most of the G. vaginalis cells attached to the vaginal epithelial cells when in combination with L. salivarius FV2 (Mastromarino et al., 2002). 2D.4 Hydrogen peroxide producing By carrying out the TMB-plus plate assay, the Lactobacillus strains were tested for their capability to produce H2O2. L. crispatus DSMZ 20584 was shown to be a strong H2O2 producing strain out of all the strains tested whilst L. acidophilus and L. reuteri were weak producers. It has been found that H2O2 also has an inhibitory effect on BV 32 bacteria and the three positive strains could be potential probiotics to be further investigated. The same study by Mastromarino also indicated that L. gasseri was H2O2 producer and inhibited growth of G. vaginalis. In the present study, the L. gasseri tested did not produce H2O2 indicating that strain variation occurs. Another study also found that L. acidophilus 48101 of vaginal origin was a H2O2 producer and shown to also inhibited G. vaginalis (McLean and Rosenstein, 2000). The L. acidophilus strain being used in this study has demonstrated potential as a probiotic for BV because of its inhibitory activity against G. vaginalis. L. acidophilus is already widely used in the dairy industry as a probiotic but there may be differences between the strains used. Further work would be required to identify the best strains for prophylaxis of BV. However, the L. acidophilus was not a strong a producer of hydrogen peroxide and it may be that all of the required probiotic characteristics cannot be found in a single strain. In that case a mixture of lactobacilli might prove to be the most effective probiotic. 33 Chapter 3 3. IMMUNE RESPONSE 3A. Introduction 3A.1 Investigation of cytokine response to Lactobacillus strains and BV bacteria These sets of experiments were carried out to investigate the nature of the cytokines produced by THP-1 macrophages and monocytic derived dendritic cells once they are exposed to BV bacteria and the Lactobacillus strains. The responses of a human monocytic cell line (THP-1), monocyte derived human dendritic cells and Langerhan’s cells (MD-DCs and MD-LCs) to potential probiotic Lactobacillus strains and BV bacteria were the focal point of these experiments. This included the following: A) Heat-killed G. vaginalis #810 and A. vaginae #38953 that are involved in bacterial vaginosis B) Heat-killed potential probiotic Lactobacillus strains: L. acidophilus, L. crispatus DSMZ 20584, L. reuteri #61.6, L. paracasei, L. rhamnosus #67B and L. gasseri #2.1. C) Mixture of L. reuteri #61.6 and L. gasseri #2.1 and BV bacteria The aim of these experiments was to determine the effect of probiotic Lactobacillus and BV associated bacteria upon THP-1 cells, MD-DCs and MD-LCs by initially measuring TNF-α production and subsequently investigating other pro-inflammatory cytokines produced. Subsequently, preliminary work was carried out using DC generated in vitro from peripheral blood monocytes (MD-DCs) using blood from healthy donors and an exploratory experiment was carried out using monocyte-derived Langerhans cells. Both of these latter cell types are representative of primary cells present in the vagina. BV as a polymicrobial syndrome as opposed to vaginitis caused by trichomonas or candidiasis does not cause inflammation. In contrast, the proinflammatory cytokine, TNF- α production has obviously been shown to increase during bacterial vaginosis in some studies. The THP-1 cell was used as a model to test the response of differentiated monocytes to Lactobacilli strains and BV bacteria and initially TNF-α production was quantified to elucidate the inhibitory effect of potential probiotics upon BV. Numerous 34 studies have indicated that some lactobacillus strains can suppress the proinflammatory reaction initiated by the immune cells in response to exposure to pathogen. Thus, the potential lactobacillus strains could be explored in depth either for the prophylaxis use or treatment of BV. 3A.2 The human monoblastic leukaemic cell line (THP-1) In view of the fact that primary tissue or peripheral blood macrophages cannot be readily propagated in vivo, various human monocytic cells lines are frequently used to model macrophage function. Their availability contributes towards the study of varying aspects of macrophage biology and biochemistry, including host defensive and secretory mechanism. In 1980, a new leukaemic cell line now known as THP-1 was obtained from a one year old Japanese boy who suffered from monocytic leukaemia. This cell line has both monocytic and immunological properties, forming loose clumps and is non-adherent (Tsuchiya et al., 1980). Upon exposure to differentiating agents such as phorbol 12myristate 13-acetate (PMA), 1, 25 dihydroxyvitamin D3 (VD3), retinoic acid cytokines TNF-alpha or IFN-gamma or the cells differentiated with the functional characteristics of mature macrophages(Tsuchiya et al., 1982),(Schwende et al., 1996), (Chen and Catharine, 2004), (Chen et al., 1996). The various agents used to induce THP-1 cells cause chromatin redistribution that resulted in a variety of morphological changes. The cells may appear either as ovoid or amoeboid, flat or ballooned, clumps or aggregated and furthermore the cell volume may also decrease due to presence of many phagocytic vacuoles and irregular nucleus (Vey et al., 1992). Another distinguishing feature is the adherence of cells to the tissue culture plastic substrate and expression of surface markers (Schwende et al., 1996) and secretion of cytokines, immunomodulation responses, expression of pathogenassociated molecular patterns (PAMPs), oncogene expression and genes involved in lipid metabolism (Auwerx, 1991). As such, the THP-1 cell line offers several major advantages as a monocytic/macrophage model system due to limitless supply and quantities for research purposes and consistent macrophage cell phenotype. Owing to these 35 characteristics, THP-1 cell line provides a valuable model to study immunomodulation responses involving cytokine production (Auwerx, 1991). 3A.3 Monocytice derived Dendritic cells (MD-DCs) and Langerhans cells (MDLCs) Dendritic cells (DCs) and Langerhans cells (LCs) are specialised sentinel immune cells known as antigen presenting cells (APC) which are essential initiators of immunity and tolerance. They form a tight cellular network of primary sentinel cells against pathogens and neoplasm and are found to be widely dispersed throughout the body. These APCs express surface molecules which recognise infectious and exogenous ligands called pattern recognition of pathogen-associated molecular patterns. These surface molecules activate APCs that have endocytosed and processed antigens so that they can prime antigen-specific T and B lymphocytes (adaptive immunity) (Gordon, 2002). They are the key cells to induction of immune responses. Langerhan’s cells are a subset of the dendritic cells and are present in the epidermis, mucosa and bronchi. They are present in the stratified squamous epithelial layer of the skin and within the submucosal lamina propria of the female reproductive tract (Zhao et al., 2003). During an infection or inflammation, the MD-DCs are recruited to the vaginal mucosa from the peripheral blood (Ilijima et al., 2007). MD-DCs are non-proliferating human cells thus there is no continuous supply as compared to the THP-1 cell line. However, these immune cells can be generated from the fresh human blood. MD-DCs can be generated in vitro from human peripheral blood mononuclear cells (PBMCs) under the influence of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) cytokines within 5-7 days (Romani et al., 1996). 3A.4 Anti-inflammatory cytokines The immunosuppresive cytokines of interest in this study are TGF-β and IL-10. TGFβ1 is a protein that that blocks the activation of lymphocytes and monocytic derived phagocytes. IL-10 is also an anti-inflammatory cytokine which is produced mainly by monocytes and known to inhibit other pro-inflammatory cytokines such as IFN-γ, IL36 12, IL-3, TNF-α and GM-CSF produced by macrophages and T-regulatory cells. The up-regulation of the immunosuppressive cytokines in exposure to the various Lactobacillus strains used in this study is an important determinant to choosing the potential probiotic which can be further investigated. 3A.5 Pro-inflammatory cytokines IL-1β is an important mediator of an inflammatory response produced by mononuclear phagocytes and other cells. This can activate macrophages and lymphocytes to produce IL-6. IL-6 is a pleitropic cytokine which can act as either pro-inflammatory or antiinflammatory cytokine also produced by macrophages in response to bacterial peptides. IL-8 is also produced by macrophage, most powerful chemo-attractant for cell mediated response to infection. Further, IL-12 is another cytokine produced by macrophages and dendritic cells in response to antigenic stimuli and selects for a pro-inflammatory response. 3A.6 Aims THP-1 cells were differentiated by artificial stimulators PMA and then challenged with the BV bacteria G. vaginalis, A. vaginae and the six potential probiotic Lactobacillus strains in varying bacterial cell density and for various time intervals. Furthermore, MD-LCs and MD-DCs were also exposed to BV organisms and the two potential probiotics, L. reuteri and L. gasseri respectively. ELISA was initially utilized to evaluate the levels of TNF- α as a preliminary screening tool followed by a more sensitive assay system, the Bio Plex (Bio-Rad), quantitatively measure TNF- α and a range of other to pro-inflammatory and anti- inflammatory molecules in the cell culture supernatants. 37 3B MATERIAL AND METHODS 3B.1 THP-1 Cell line THP-1 cells were obtained from the Dr Merilyn Hibma, Virology Research Unit of the University of Otago Microbiology and Immunology Department. THP-1 cells served as a constant supply of monocytes/macrophage for the cytokine study. The cells were cryopreserved in liquid nitrogen at a concentration of 5x106 cell/ml in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% foetal calf serum (FCS) and 10% Dimethyl Sulfoxide (DMSO). 3B.2 Resuscitation and subculture of THP-1 cells The instructions recommended for growth and maintenance of THP-1 cells were obtained from the ATCC website. However, for this study, the THP-1 cells were routinely propagated in DMEM rather than Roswell Park Memorial Institute-synthetic tissue culture (RPMI) medium, supplemented with 10% heat inactivated FCS, 0.05 mM 2-Mercaptoethanol (2ME), penicillin 100 U/ml and 100 ug/ml of streptomycin (Gibco/BRL Life Technologies). The use of DMEM was due to its high nutrient and glucose content which supports cell viability of many cell lines (Wu et al., 2009). For resuscitating THP-1 cells, the vial was thawed and the cells were immediately transferred drop wise into a 15 cm2 cell culture flask (BD) with 5 ml of DMEM supplemented with 10% FCS and antibiotics. Following this, the flask was incubated at 37 °C in a humidified 5 % CO2 atmosphere for 5 days and culture medium replaced after day 5. The cells were sub-cultured when the total cell concentration reached 1x106 cells/ml by seeding new flasks with 3x105 cells/ml and adding 12 ml of growth medium. To maintain the cells at 8x105 – 1x106 cell/ml, the THP-1 cells were split on every 4th day. The trypan blue (BD) exclusion method was carried out regularly to assess viability and cells were counted using a haemocytometer. Furthermore, THP-1 cell passages were maintained below 15 to avoid the cells undergoing abnormal cell character alterations. More than 25 cell passages have been reported to give inaccurate results due to hyperproliferation of the cells themselves (Kremlev et al., 1997). 38 3B.3 Preparation of PMA The lyophilised PMA agent (SIGMA) was reconstituted to stock solution of 1 mg/ml according to the manufacturer’s instruction and stored as 20 ul aliquots in Eppendorf tubes at -20 °C. 3B.4 Preparation LPS and LTA LPS from E. coli 055:B4 strain (SIGMA) was received as 5 mg lyophilised powder and reconstituted with 5 ml of sterile water to a stock solution concentration of 1 mg/ml. This was dispensed as aliquots of 50 ul in Eppendorf tubes. Purified LTA from Stapylococcus aureus strain (SIGMA) was also received as 5 mg lyophilised powder and reconstituted with 5 ml sterile water to give stock concentration of 1mg/ml. Aliquots of 50 ul of LPS and LTA in Eppendorf tubes were then stored at -20 C. 3B.5 Optimising PMA dose to differentiate THP-1 cells from monocytes to macrophage The THP-1 cells were propagated in 300 cm2 tissue culture flasks (Nunc), aseptically transferred to 50 ml falcon tubes (BD) and centrifuged at 300 g for 5 minutes and supernatants discarded. The pellets were then re-suspended with fresh DMEM supplemented with 1% FCS, 0.05 mM 2-ME and 100 U/ml penicillin and 100 ug/ml streptomycin adjusted to 2x106 cells/ml as stock. The THP-1 cells were then further diluted to 1x106 cells/ml in 200 ul aliquots and treated with different doses of PMA; 20 ng/ml, 30 ng/ml and 40 ng/ml respectively. THP-1 cells treated with various PMA doses were dispensed in 200 ul/well in a flat bottom 96-well plated (BD). Each PMA dose was done in triplicate with 3 sets of experiments each for 24, 48 and 72 h. The plates were then incubated at 37°C with 5% CO2 over different incubation times, 24, 48 and 72 h, during which each plate was checked microscopically for morphological appearances of the cells. The negative control was 5x105 cells/ml with no differentiating agent processed under the same condition as for the tests. The same procedure was then performed to test lower dose of PMA taken to differentiate the THP-1 cells over a 24 h period. PMA dose of 20 ng/ml, 10 ng/ml and 5 ng/ml was used in the manner as above. 39 3B.6 Tests to confirm differentiation of THP-1 cell The viability of adherent, differentiated THP-1 macrophage cells was measured on the plate reader (Tecan) at the OD of 595 nm after staining the cell cultures with 1% crystal violet. The viable cell count technique was also used to count viable THP-1 cells that were differentiated. The two methods are described as follows. 3B.6.1 Crystal violet staining Crystal violet staining of microtitre plate cultures was used to assess the proportion of differentiated (attached) THP-1 cells. The washing steps remove undifferentiated cells leaving the differentiated cells only in the plate. Crystal violet is used to stain the differentiated cells so that the amount retained in each microtitre plate well is proportional to the number of cells. After observation of the plates under microscopy to determine the differentiation of THP-1 cells, the supernatant fluid was gently flicked out into a container of disinfectant. The cells were washed twice by adding three drops of pre-warmed sterile PBS into each well using a sterile dropper and gently flicking this out as above. A volume of 100 ul of methanol was added to each well, the plate was incubated at room temperature for 60 minutes, flicked out as above and then left on absorbent paper to air dry. Crystal violet (1% strength) in a 100 ul volume was added into each well and incubated at room temperature for 5 minutes followed by washing step as above. A volume of 100 ul/well of 33% glacial acetic acid was added to each well and each well gently thoroughly mixed with a new pipette tip followed by absorbance reading at OD of 595nm on the plate reader. The experiments included triplicate tests with 3 experiments carried out and the average of the absorbance recorded. 3B.6.2 Viable count of THP-1cells by trypan blue exclusion The THP-1 cell supernatant was gently flicked out into a container of disinfectant and cells washed twice by adding three drops of pre-warmed sterile PBS into each well using a sterile dropper and gently flicking this out. Trypsin stock at 3x concentration was diluted to 1x in sterile PBS with 50 ul added to each well and incubated at 37 °C for 7-10 minutes during which the cells were observed under high power microscope (100x objective) to confirm detachment of cells from the bottom of the wells. This was followed by addition of 150 ul of DMEM (supplemented with 10% FCS, 2ME and 40 antibiotics) into each well and 10 ul was added to equal volume of trypan blue stain in an Eppendorf tube and mixed thoroughly. Finally, 10 ul of stained cells were pipetted onto a haemacytometer and live and dead cells were counted. The average from all triplicates over 3 experiments was recorded. 3B.7 Optimising THP-1 cell concentration To determine the optimal number of THP-1 cells that would differentiate with 20 ng/ml of PMA after 48h, THP-1 cells were seeded from 5x106- 1x104 cells/ml with 200 ul added into appropriated wells in 2 sets. After 48 h one set of cells were enumerated for viability using the trypan blue exclusion whilst the other set underwent the crystal violet procedure as above. The experiments included triplicate tests per sample with 3 experiments and average recorded. 3B.8 Optimising dose of LPS and LTA To optimise the dose of LPS or LTA used to stimulate the production of cytokines from THP-1 cells, supernatants from the cells treated with these two reagents were analysed for the presence of TNF-α. The THP-1 cells were differentiated with 20 ng/ml of PMA, dose range of 0.5, 1.0 and 2.0 ug/ml of LPS and LTA were added into appropriate wells including negative control consisted of DMEM medium alone and incubated for 24 h at 37 °C + 5% CO2 followed by collection of supernatants into Eppendorf tubes and stored at -20 °C for TNF-α measurement using ELISA assay. 3B.9 Optimising lactobacilli and BV bacteria concentration on THP-1 cells To determine the optimum dose of bacterial load that would trigger high level of TNFα level 1x106 cells/ml of THP-1 cells differentiated for 48 h with 20 ng/ml of PMA were challenged with various doses of A. vaginae, G. vaginalis, L. reuteri and L. gasseri. The doses ranging from 5x108 - 5x102 bacterial cells/ml, were added in 200 ul volumes to appropriate wells and incubated at 37 °C + 5% CO2 incubator for 24 h followed by collection of 150 ul of supernatant from each well for storage in sterile Eppendorf tubes for subsequent cytokine assay. The LPS and LTA were added at 10 ng/ml in 200 ul/well volumes and the negative control was DMEM medium alone. The tests and controls were done in triplicate in 3 sets of experiments. 41 3B.10 Assessment of cytokine responses of differentiated THP-1 cells following exposure to bacteria THP-1 cells were seeded at 2.5 x106 cells/ml and differentiated with 20ng/ml of PMA for 48 h incubation at 37 C+5% CO2. The Lactobacillus strains were adjusted to 5x108cfu/ml, A. vaginae to 5x107 cfu/ml and G. vaginalis to 5x108 cfu/ml and these were added in 1 ml volumes to stimulate the differentiated THP-1 cells for 24 h as described above. Supernatants from triplicate tests per sample were pooled in Eppendorf tubes and stored in -20°C for subsequent cytokine analyses. The experiment was repeated three times including LPS, LTA and negative control (DMEM medium with no bacterial cells). 3B.11 Culture of human peripheral blood monocytes PBMCs were extracted from 80 ml of whole blood from five healthy female volunteers using the Ficoll Paque density centrifugation method. CD14+ monocytes purified from the PBMC were selected using the anti-CD14 magnetic beads assay as described in a previous study (Geissmann et al., 1998). The Ethics committee of the University of Otago approved this study (Permit F10/004) and subjects were informed and gave written consent. The growth medium used was RPMI 1640 supplemented with 2 mM 1-glutamine, 10% heat-inactivated FCS, and 100 U/ml penicillin + 100ug/ml streptomycin (Gibco/BRL Life Technologies. The procedures associated with the generation of MD-DCs (see below) were all carried out by Ms Michelle Wilson, Manager, Immunology Laboratory. 3B.11.1 Harvesting blood monocytes Human blood collected from each donor was processed separately. Whole blood was re-suspended in DPBS in equal portions to a total volume of 40 ml in Falcon (BD) tubes. Followed by gradual layering of 26 ml of blood onto a 15ml Ficol paque PLUS in sterile 50 ml Falcon tube and centrifuged at 800 rcf for 20 minutes at room temperature. The buffy coat containing leucocytes was then removed from each tube and transferred into a 50 ml Falcon tube, washed 3x with sterile DPBS and centrifuged at 200 rcf for 15 minutes at room temperature. The cells were then re-suspended in 1ml 42 cold MACS buffer and cell yield counted by trypan blue exclusion. Leucocytes (1x107 cells) were then resuspended in 80 ul of cold MACS buffer, 20 ul of huCD14 magnetic MACS beads (Miltenyibiotec) were added and the suspension was incubated at 4 °C for 15 minutes. The cells were then spun down and washed with 20x volume in cold MACS buffer and resuspended in 500 ul MACS buffer at 1x108 cells. Positive immunomagnetic selection of CD14+ monocytes was carried out using the AutoMACS (Miltenybiotec) following the manufacturer’s instructions. 3B.11.2 Generating dendritic cells from CD+14 monocytes Monocytes) were washed with 3x volume MACS buffer and re-suspended in 1 ml RPMI1640 +containing 2 mM L-glutamine, penicillin/streptomycin, 10% FCS and 25 ng/ml huGM-CSF and IL-4. The monocytes at 1x106 cells/ml were re-suspended in RPMI 1640 medium (recipe above) and incubated at 37 C + 5% CO2 for 6 days. The cells were fed by replacing half the medium with fresh medium containing the cytokines on day 3.The huMD- DCs were then harvested on day 6 (Romani et al., 1994), (Geissmann et al., 1998). 3B.12 MD-DCs challenge with BV bacteria and lactobacilli strains To test for the response of MD-DCs to exposure to BV bacteria alone or in combination with L. reuteri or L. gasseri, 200 bacterial cells (2x108 cfu/ml) per dendritic cell was the ratio of bacteria to cells used. The MD-DCs and the heat killed bacterial cells were incubated in total volume of 250 ul at 37 °C with 5% CO2 for 24 h and 170 ul supernatant were collected for TNF-α and other cytokines assay. 3B.13 TNF- α cytokine ELISA Supernatants of differentiated THP-1 cells and MD-DCs treated with LPS, LTA, killed BV and lactobacilli bacterial extracts were tested for TNF-α using a quantitative solid phase sandwich enzyme immunoassay (BD). The plate wells were coated with 50 ul of purified anti-mouse cytokine TNF-α antibody diluted in phosphate coating buffer (Appendix for recipe) to a final concentration of 2 ug/ml and incubated at 37°C for 1 h. The plates were washed 6 times with wash buffer (1x PBS containing 0.05% Tween 20), blocked with 200 ul/well of Blocker Buffer (1% 43 Bovine serum albumin [BSA] in 1x PBS), incubated at 37 C for 1 h and washed again. Volumes of 100 ul/well of sample, blank and standard (doubling dilution of the recombinant TNF in blocker buffer from 2000-15 pg/ml) was added to the plates which were incubated at 37 °C for 2 h followed by washing step as above. The wells were then treated with 100 ul of biotinylated mouse anti-human TNF detection antibody (diluted in blocker buffer to a concentration of 1 ug/ml) and incubated at 37 °C for 30 minutes. The plates were washed as above and treated with 100 ul/ml of streptavidinhorseradish peroxidase conjugate for 20 minutes at 37 °C and washed again as above. The tetramethylbenzidine dihydrochloride (TMB) substrate was added to develop colour reaction (100 ul per well) and stopped with 100 ul per well 1NH2SO4 followed by optical density measurement readings at 450nm. A standard curve was automatically generated from the recombinant human TNF by the immunoassay spectrophotometer instrument (Microplate Manager Bio-Rad Laboratories, Inc) and manually corrected to create a linear standard curve. The average absorbance readings of each sample were then calculated from the standard curve. The samples with values over the highest point on the standard were then diluted 1:10 or 1:20 and repeated as above. 3B.14 Bio-Plex assay for cytokines IL-1b, IL-6, IL-8, IL-10 and IL-12 The Bio-Plex assay kits were purchased from Bio Rad for assay of multiple cytokines and single cytokine TGF-β1 and performed according to manufacturer’s instructions. 3B.14.1 Principle of assay The bioplex assay for the above cytokines was carried out using the MILLIPEX MAP kit. The principle for the bioplex assay for cytokines is based on luminex MAP technology using multiple conjugated fluorescent-coded beads known as microspheres. Each bead set is coated with specific capture antibody to which the test sample is introduced, followed by addition of biotinylated detection antibody and Streptavidin-PE conjugate as the reporter molecule on surface of each microsphere. The microspheres are then passed through a set of lasers. The first laser excites the internal dye whilst the second laser excites PE, the fluorescent dye on the reporter molecule which was read at high-speed digital-signal processors. Finally each individual microsphere is quantified and results obtained are based on fluorescent reporter signals. 3B.14.2 Preparation of reagents for immunoassay 44 3B.14.2.1 Preparation of antibody immobilized beads Individual vials of antibody-immobilized beads were sonicated for 30 seconds and mixed by vortex for 60 seconds. From each antibody bead vial, 60 ul was added to a mixing bottle and topped up to 3 ml with bead diluents and mixed well by vortex. 3B.14.2.2 Preparation of quality control and wash buffer Quality control 1 and 2 were reconstituted with 250 ul of deionised water, mixed thoroughly on the vortex, incubated at room temperature for 5-10 minutes and transferred to labelled polypropylene microfuge tubes. To prepare 1x wash buffer, 30 ml of 10x wash buffer was diluted with 270 ml deionised water and brought to room temperature before use following the manufacturer’s instructions. 3B.14.2.3 Preparation of human cytokine standard The human cytokine cocktail standard was reconstituted with 250 ul of deionised water to a concentration of 10 000 pg/ml. The vial was mixed several times, vortexed for 10 seconds and allowed to incubate at room temperature for 5-10 minutes before being transferred to polypropylene microfuge tube. The cytokine standard was then serially diluted from 10,000-3.2 pg/ml (50 ul of standard to 200 ul of assay buffer) and assay buffer alone was used as the background or blank. 3B.14.2.4 Bio-Plex immunoassay procedure The microtitre filter plate was pre-wet with 200 ul/well of assay buffer, sealed and mixed on plate shaker for 10 minutes at room temperature followed by removal of assay buffer by vacuum. The standards, controls and background were added to appropriate wells (25 ul/well) followed by addition of assay buffer (25 ul/well) to all the wells. A volume of 25 ul from each sample was added to appropriate wells and 25 ul of DMEM (culture medium as matrix) to background, standards and control wells. The premixed antibody beads were added at 25 ul/well to all wells, sealed with a plate sealer and incubated overnight at 4 °C. This was followed by removal of fluid by vacuum and washing with 200 ul/well of wash buffer twice with vacuum filtration between each wash. The next step was addition of 25 ul/well of detection antibodies, sealing of the microtitre plate with a plate sealer and incubation with agitation on a plate shaker for 1 hr at room temperature. The microtitre plate was sealed with a further 45 agitation on the plate shaker at room temperature for 30 minutes after addition of streptavidin-phycoerythrin (25 ul/well). The washing and vacuum steps were repeated as above with further agitation on the plate shaker at room temperature for 5 minutes after the addition of sheath fluid to all the wells. Finally, the microtitre plate was run on the Luminex immunoassay autoanalyser. The Luminex was operated by Ms Claire Fitzpatrick. 3B.15 Bio-Plex assay for cytokine TGF-β1 The principle for this cytokine assay is the same as for the multiplex, however only a single cytokine is measured according to manufacturer’s instructions. 3B.15.1 Preparation of reagents for immunoassay 3B.15.1.2 Treatment of cell culture supernatants containing serum In order to measure activated TGF-β1, it is necessary to treat the supernatants with acid purposely to remove the pro-region peptide (also known as latency associated peptide) which occurs at low pH. For each of the samples, 25 ul was transferred to microfuge tubes and treated with 2 ul of 1.0 N HCl, mixed and incubated with agitation on a plate shaker for 15 minutes at room temperature. The acid-treated, serum-containing cell culture supernatants were then neutralised with 2 ul of 1.0 N NaOH before the TGF-β1 assay. 3B.15.1.3 Bio-plex immunoassay procedure for TGF-β1 The singleplex immunoassay procedure for TGF- β1 was carried out in a similar stepwise manner as the one carried out for the multiplex as described above. 46 3C. RESULTS 3C.1 Determining the PMA dose to differentiate THP-1 cells from monocytes to macrophage (preliminary experiments) Initial experiments were carried out to determine the optimal dose of PMA to differentiate the THP-1 cells from the precursor stage of monocytes to macrophages. 3C.1.1 Crystal violet staining and Absorbance reading Figure 1.0 (a) presents the crystal violet staining of the adherent differentiated THP-1 macrophages after exposure to 20, 30 and 40 ng/ml of PMA for 24, 48 and 72 hr incubation periods. Although PMA at 40ng/ml for 72 hr gave a higher absorbance reading of more than 1.2, similar absorbance values for all three concentrations of PMA occurred after 48 hr which suggested that this may represent a plateau. Therefore the dose was titrated and the cells were again exposed for 48 hr. Figure 1.0 (b) indicates that both 20 ng/ml and 5ng/ml were able to differentiate the cells with no clear difference in the absorbance values (0.391 and 0.395). 3C.1.2 Viable cell count by trypan blue As seen from Figure 1.0 (c), the results of viable cell count using trypan blue indicated that treatment with 20 ng/ml of PMA yielded 7.56 x105 viable cells and 5 ng/ml of PMA yielded 8.28 x105 viable cells. Surprisingly, a trend towards higher cell death was noted at 5ng/ml so the dose of 20 ng/ml of PMA for 48 hr was taken as the optimal dose and time to differentiate the THP-1 cells for subsequent experiments. 3C.1.3 Determining the THP-1 cell concentration It was also necessary to optimise the THP-1 cell concentration needed to produce the optimal cytokines upon stimulation by heat inactivated lactobacilli and BV bacteria, LPS and LTA. Using crystal violet staining it can be seen from Figure 2.0 that the THP-1 cells seeded at 5x106 cells/ml and 1x106 cells/ml differentiated with 20 ng/ml of PMA yielded more adherent cells as indicated by the high absorbance reading. There is a clear trend 47 towards decreasing yield of THP-1 cells when the seeding concentration was reduced from 5x105 to 1x104 cells/ml. This preliminary analysis indicated the THP-1 cells seeded at either 5x106 or 1x106 cells/ml would be optimal. 3C.1.4 Lactobacilli and BV bacteria concentration on THP-1 cells for the generation of TNF-α In order to assess the optimal bacterial load needed to trigger TNF-α production by the THP-1 cells a further ‘optimisation’ experiment was also necessary. As shown from the graph on Figure 4.0, bacterial load of 5x108 cfu/ml for L. reuteri, L. gasseri and A. vaginae triggered high TNF-α. Lower concentrations of cells demonstrated greatly reduced TNF-α production in response to all bacterial strains extracts. LTA and LPS were taken as positive controls for Gram positive and Gram negative bacterial cell components respectively. A dose of 5x108 cfu/ml was required to stimulate TNF-a production to these stimuli. The negative control (DMEM media alone) did not trigger much TNF-α as expected. A bacterial load of 5x108 cfu/ml was therefore chosen for subsequent experiments. 48 1.4 Absorbance 595 nm 1.2 1 0.8 0.6 24 hr 0.4 48 hr 0.2 72 hr 0 20 ng/ml 30ng/ml 40ng/ml Nega9ve control PMA in ng/ml Figure 1.0 (a) Absorbance (OD595nm) of THP-1 cells at 1x106 cells/ml differentiated with 20, 30 and 40 ng/ml of PMA over 24, 48 and 72 hr followed by crystal violet staining and absorbance reading to determine the degree of differentiation indicated by the uptake of the crystal violet stain by adherent cells. 49 0.45 0.4 Absorbance at 595nm 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 20ng/ml 10ng/ml 5ng/ml negative control PMA Figure 1.0 (b) Absorbance (OD 595nm) of THP-1 cells at 1x106 cells/ml after 48 hr of differentiation with 20, 10, and 5 ng/ml of PMA followed by crystal violet staining and absorbance reading to determine the degree of differentiation indicated by the uptake of the crystal violet stain by adherent cells. 50 900000 800000 700000 Number of cells 600000 500000 Live cells 400000 Dead cells 300000 200000 100000 0 20 10 5 Negative PMA concentration (ng/ml) Figure 1.0 (c) Viable cell count of THP-1 cells at 1x106 cells/ml after 48 hr differentiation with 20, 10, 5 ng/ml of PMA followed by trypan blue exclusion method to determine live and dead cells. 51 0.9 Absorbance 595nm 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 5x1000000 1x1000000 5x100000 1x100000 5x10000 1x10000 THP‐1 cell concentra9on (cells/ml) Figure 2.0 Absorbance measurement (OD595nm) of THP-1 cells seeded at various concentrations after differentiated by 20 ng/ml of PMA for 48 hr followed by crystal violet staining to determine the degree of differentiation indicated by the uptake of the crystal violet stain by adherent cells. 52 4000 3500 TNF‐alpha pg/ml 3000 2500 2000 1500 5x100000000 cells/ml 5x1000000cells/ml 5x10000cells/ml 1000 5x100 cells/ml 500 0 Figure 3.0 TNF-α production by differentiated THP-1 cells at 1x106 cells/ml after exposure to various concentrations of L. reuteri #61.6, L. gasseri #2.1, G. vaginalis #810 and A. vaginae #38953 and measured by ELISA. Each experiment consisted of three tests with pooled supernatants assayed and an average of 3 experiments. Positive controls: LPS and LTA (10 ng/ml for each); negative control: medium only. 53 3C.2 ELISA assay for TNF-α production by THP-1 cells stimulated by Lactobacillus strains and BV bacteria Figure 4.0 (a) presents individual TNF-alpha produced by THP-1 cells in response to stimulation by Lactobacillus strains and BV bacteria from three main experiments. There is significant variance between each set of experiments as seen from the results. This could be due to several reasons such as pipetting error, discrepancies in the bacterial cell counts and dilutions and alterations in THP-1 cells after several passages. Figure 4.0 (b) presents the average results (in error bars) obtained from the three main ELISA assay of TNF-alpha produced by THP-1 cells in response to stimulation by Lactobacillus strains and the BV bacteria. In these experiments certain trends were observed in the production of TNF-α. Out of all the lactobacilli, L. rhamnosus triggered least TNF-alpha (1492.8 pg/ml) while L. crispatus triggered the highest levels of TNFalpha (4992.5 pg/ml). On the other hand, of all the bacteria tested, G. vaginalis and LTA triggered the least amount of TNF-alpha (1028.2pg/ml). For the combined bacteria, L. gasseri + A. vaginae triggered the highest TNF-alpha with 6098.5 pg/ml whilst L. reuteri + G. vaginalis with 2585.7 pg/ml triggered the lowest. For L. rhamnosus, L. paracaei and L. gasseri, the combination of lactobacilli bacteria and either of the BV bacteria however, resulted in an increase in the amount of TNF-α produced. This was an additive effect, rather than synergistic effect. 3C.3 Preliminary experiments to determine TNF-α production from human MDDCs and human MD-LCs by ELISA assay In order to test whether human MD-DCs and MD-LCs would produce TNF-α upon exposure to lactobacilli and BV bacteria, preliminary experiments were necessary. Figure 5.0 (a) shows the preliminary results of TNF-α produced by human donor P1 MD-DCs stimulated with L. reuteri, L. reuteri + G. vaginalis and LPS. The combination of L. reuteri + G. vaginalis triggered slightly more TNF-α (1528.1 pg/ml) than L. reuteri (1398.8 pg/ml) and LPS. 54 Figure 5.0 (b) presents the preliminary results of TNF-α production from donor P2 MDLCs in response to L. reuteri, L. gasseri and BV bacteria (G. vaginalis + A. vaginae).The highest TNF-α was triggered by combination of L. gasseri+ BV bacteria (G. vaginalis + A. vaginae) with 3059.6 pg/ml whilst no TNF-α was recorded upon L. reuteri exposure. It is possible that the obvious big contrast in TNF-α production by the combination of L. gasseri + BV bacteria was attributable to extraneous bacterial contamination. 3C.4 TNF-α production by MD-DCs from three different female donors measured by ELISA Figure 6.0 (a),(b) and (c) presents ELISA assay results of TNF-α level from three different donors MD-DCs response to L. reuteri, L. gasseri, BV bacteria (G. vaginalis + A. vaginae) and LPS. Figure 6.0 (a) shows results for donor 1 shows that the TNF-α level triggered by BV bacterial (3589.2pg/ml) was lower than in combination with lactobacilli. The combination of L. reuteri + BV (4842.8 pg/ml) and the L. gasseri + BV (6209 pg/ml) has indicated an additive response. Interestingly, L. reuteri triggered less TNF-α (925.2 pg/ml) than L. gasseri (2079.3 pg/ml). Figure 6.0 (b) results for donor 2 shows that a combination of L. gasseri + BV bacteria triggered higher TNF-α production (3751.1 pg/ml) than the sum of the TNF-a when the bacteria were tested alone. This shows a synergistic effect of L. gasseri onto the BV bacteria. By contrast, the combination of L. reuteri + BV bacteria resulted in the production of 774.3 pg/ml TNF-a. The combination L. gasseri + A. vaginae produced much higher TNF-α (2777.2 pg/ml) than L. gasseri +G. vaginalis Figure 6.0 (c) presents TNF-α results from donor 3 MD-DCs. The BV bacteria triggered a TNF-α production of 1079.1 pg/ml as compared to L. gasseri + BV bacteria with 3762.6 pg/ml, which is, again, indicative of a synergistic effect. This was also seen with the L. gasseri + A. vaginae similar (3758.pg/ml). Most interestingly, L. reuteri appeared to have an inhibitory effect on BV. Since these are preliminary results, further work is required to determine whether this result is reproducible. 55 L.acido+G.vaginalis L.acido+A.vaginae L.reuteri L.reut+G.vaginalis L.reut+A.vaginae L.crispatus L.crisp+G.vaginalis L.crisp+A.vaginae L.rhamnosus L.rham+G.vaginalis L.rham+A.vaginae L.paracasei L.parac+G.vaginalis L.para+A.vaginae L.gasseri L.gas+G.vaginalis L.gas+A.vaginae G.vaginalis 12000 10000 8000 6000 4000 2000 0 L.acidophilus L.acidophilus L.acido+G.vaginalis L.acido+A.vaginae L.reuteri L.reut+G.vaginalis L.reut+A.vaginae L.crispatus L.crisp+G.vaginalis L.crisp+A.vaginae L.rhamnosus L.rham+G.vaginalis L.rham+A.vaginae L.paracasei L.parac+G.vaginalis L.para+A.vaginae L.gasseri L.gas+G.vaginalis L.gas+A.vaginae G.vaginalis A.vaginae LPS LTA Nega9ve control A.vaginae LPS Expt 1 Expt 2 Nega9ve control Expt 3 LTA a b TNF‐alpha (pg/ml) 7000 6000 5000 4000 3000 2000 1000 0 Figure 4.0(a) The first figure shows three sets of experiments presenting the TNF-α production by THP-1 macrophages in response to Lactobacillus strains and BV bacteria 56 by ELISA. (b) The second figure shows the average results of the 3 main experiments shown in error bars. THP-1 cells at 2.5x106 cells/ml differentiated with 20 ng/ml of PMA for 48 hr exposed to 5x108 cfu/ml Lactobacillus strains, 1x107 cfu/ml of A. vaginae and 5x108 cfu/ml of G. vaginalis. TNF alpha (pg/ml) a TNF‐alpha (pg/ml) 2000 1500 1000 500 0 L.reuteri L.reut+G.vag LPS Neg. control TNF alpha (pg/ml) b 3500 3000 2500 2000 1500 1000 500 0 L.reuteri L.gasseri BV L.reut+BV L.gas+BV Figure 5.0 (a) The first figure shows the preliminary ELISA assay results of TNFalpha levels from female donor P1 MD-DCs stimulated with L. reuteri, G. vaginalis and LPS. (b)The second figure presents values of TNF-α production by donor P2 MDLCs after exposure to L. reuteri, L. gasseri and BV bacteria (G. vaginalis + A. vaginae). No LPS was added in donor P2 because of insufficient MD-DCs. Monocytes (CD14+) harvested from human blood were exposed to cytokines GM-CSF and IL-4 to generate MD-DCs and GM-CSF and were later exposed to lactobacilli and BV bacteria. 57 TNF-alpha pg/ml a 8000 6000 4000 2000 0 TNF-alpha pg/ml b 4000 3000 2000 1000 0 TNF-alpha pg/ml c 4000 3000 2000 1000 0 Figure 6.0 ELISA assay of TNF-α level from MD-DCs from three different donors after exposure to L. reuteri, L. gasseri, G. vaginalis, A. vaginae, BV bacteria (G. vaginalis + A. vaginae) and LPS.(a) Donor 1,(b) Donor 2 and (c) Donor 3., Monocytes (CD14+) were isolated and exposed to cytokines GM-CSF and IL-4 to generate MDDCs. 58 3C.5 Bio-Plex immunoassay results In order to investigate a range of cytokines induced in THP-1 cells and MD-DCs in response to BV and a potentially moderating effect of lactobacilli, a bioplex suspension array assay was employed. Both pro-inflammatory and anti-inflammatory cytokines were quantified using this sensitive and accurate system. Measurement of immunosuppressive cytokines TGF- β1 and IL-10 and pro-inflammatory cytokines IL1β, IL-6, IL-8, IL-12 and TNF-α was carried out on cell supernatants from both the THP-1 cells and MD-DCs. 3C.5.1.Anti-inflammatory cytokines 3C.5.1.1 TGF-β1produced by THP-1 Figure 7.0 presents the results from bioplex assay for TGF-β1 production by THP-1 cells. There is a large variation between the TGF-β1 levels in all three experiments. However all the values are below 100 pg/ml and the negative control suggests that THP-1 cells spontaneously produce TGF-β1. On the basis of these results it could be said that most of the Lactobacilli actually suppress TGF-β1 production. The exception is in Experiment 3 where L. rhamnosus tends to increase TGF-β1 when used in combination with G. vaginalis and A. vaginae. In experiment 1 & 2 the values are probably too low to be considered functionally significant. 3C.5.1.2 IL-10 produced by THP-1 Figure 8.0 shows IL-10 production by THP-1 cells in response to lactobacilli and BV bacteria. Again, there was variation in IL-10 levels between each of the 3 experiments which prevented detection of statistical significance on the combined data from all 3 experiments. A combination of BV bacteria and lactobacilli appeared to trigger more IL-10 than single bacterial exposure as seen for the L. gasseri and A. vaginae and L. rhamnosus and G. vaginalis. For experiment 1, a trend towards more IL-10 production occurred when lactobacilli were combined with G. vaginalis than lactobacilli combined with A. vaginae. The trend showed that a combination of lactobacilli with BV bacteria triggered high levels of IL-10. The negative control for all 3 experiments produced the least IL-10. 59 These results could suggest that the equilibrium between pro-inflammatory cytokine and anti-inflammatory cytokine production to BV bacteria may be changed in favour of anti-inflammatory responses when lactobacilli are present. 3C.5.2.Pro-inflammatory cytokines 3C.5.2.1 IL-1β produced by THP-1 Figure 9.0 shows the IL-1B produced by THP-1 cells, (a) results from 3 separate experiments and (b) the average result from the 3 experiments presented in error bars. It is obvious that, again, the results vary between each experiment. No difference is discernable between individual BV bacteria alone compared with the same bacteria in combination with lactobacilli. 3C.5.2.2 IL-6 produced by THP-1 The IL-6 produced by THP-1 cells ranged from 18701 pg/ml (L. gasseri experiment 3) to 29207 pg/ml (G. vaginalis experiment 3).Combinations of bacteria seemed to produce more of IL-6 than single bacteria exposure (see appendix D). However, this graph suggests that the amount of IL-6 in the supernatants exceeds the maximum amount detectable in the assay system so it is impossible to make accurate comparisons. These supernatants would have needed to be diluted to gain accurate quantification of IL-6. 3C.5.2.3 IL-8 produced by THP-1 The results of IL-8 levels produced by THP-1 cells after exposure to both single bacteria and in combination. Interestingly the negative control (DMEM medium only) produced high levels of IL-8. The IL-8 produced ranged from 27103 pg/ml (L. paracasei + A. vaginae) to 29022 pg/ml (L. rhamnosus) between all the 3 experiments (see appendix D). However, the very high levels of IL-8 in the negative controls obscured any differences in the test samples. Again, titration of the samples would be required to detect any differences in IL-8 in the various samples. 3C.5.2.4 IL-12 produced by THP-1 As shown from the graph on Figure 10.0, IL-12 results ranged from 0 pg/ml (negative control) to 5 pg/ml (L. paracasei + G. vaginalis) for experiment 1. IL-12 levels from 60 experiment 2 ranged from 1 pg/ml (negative control) to 15 pg/ml (L. crispatus) and 1 pg/ml (negative control) to 21 pg/ml for experiment 3. These levels were regarded as too low to be functionally significant. 3C.5.2.5 TNF-α produced by THP-1 TNF-α produced by the THP-1 cells in response to the lactobacilli and BV bacteria in all the 3 experiments was above 20000 pg/ml with negative control ranging from 1357.5 to 8518.5 pg/ml between the 3 experiments (see appendix 4). The amount of cytokine in the supernatant has exceeded the level calculable by the assay system. This has indicated the sensitivity of bioplex as compared to ELISA where the amount of TNF-α detected was approximately 9500 pg/ml with the most above 3000pg/ml using the same supernatant. Again, a titration of supernatants would be required to discern differences. 61 100 90 80 Lacido+Avag Lreut+Avag Lcrisp+Avag Lpara+Avag Lgas+Avag Lrham+Avag 70 Expt 1 Lacido+Gvag Lreut+Gvag Lcrisp+Gvag Lpara+Gvag Lgas+Gvag Lrham+Gvag 60 40 Expt 2 Lacido Lreut Lcrisp Lpara Lgas L.rham Gvag Avag LPS LTA Neg control 50 30 Expt 3 62 20 10 0 Figure 7.0 Bioplex assay for TGF-beta 1 levels by THP-1 cells in response to lactobacilli and BV bacterial extracts TGF‐beta (pg/ml) 7000 6000 Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 5000 L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag 4000 3000 2000 L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control Expt 1 Expt 2 Expt 3 L.acido+A.vag L.reut+A.vag L.crisp+A.vag L.para+A.vag L.gas+A.vag L.rham+A.vag 1000 L.acido+G.vag L.reut+G.vag L.crisp+G.vag L.para+G.vag L.gas+G.vag L.rham+G.vag 0 L.acidophilus L.reuteri L.crispatus L.paracasei L.gasseri L.rhamnosus G.vaginalis A.vaginae LPS LTA Neg control a b IL‐10 (pg/ml) 4500 4000 3500 3000 2500 2000 1500 1000 500 0 63 Figure 8.0 Bioplex assay for IL-10 levels by THP-1 cells in response to lactobacilli and BV bacterial extracts presented as 3 separate experiments (a) and in average (b). IL‐10 (pg/ml) Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 25000 Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 20000 L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag 15000 5000 0 10000 b 25000 L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control a IL‐1B (pg/ml) 20000 15000 10000 5000 0 Figure 9.0 Bioplex assay for IL-1β produced by THP-1 cells in response to lactobacilli Expt 1 Expt 2 average Expt 3 64 and BV bacterial extracts. (a) IL-1β values from 3 experiments and (b) the average and error bars of the combined 3 experiments. IL‐1b (pg/ml) Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 25 L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag 20 L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control 15 10 5 0 Expt 1 Expt 2 65 Expt 3 Figure 10.0 Bioplex assay for IL-12 levels by THP-1 cells in response to lactobacilli and BV bacterial extracts. IL‐12 (pg/ml) 3C.6 Preliminary Bio-Plex assays using supernatants from MD-DCs 3C.6.1 Anti-inflammatory cytokines 3C.6.1.1 TGF-β1produced by MD-DCs Figure 11.0 shows TGF-β1 levels produced by MD-DCs from 2 different donors in response to exposure to lactobacilli and BV bacterial extracts. These results suggested that the TGF-β1 levels produced in response to combination of L. reuteri and G. vaginalis were the highest but again, reflecting the results obtained with the THP-I cells, the production of this cytokine appeared to highest in the negative control. 3C.6.1.2 IL-10 produced by MD-DCs Figure 12.0 presents the IL-10 produced by the MD-DCs from 2 donors in response to lactobacilli and BV bacteria. Donor 3 triggered more IL-10 in response to combined bacteria (L. gasseri + A. vaginae, 2703 pg/ml and L. reuteri + A. vaginae, 1297.3 pg/ml) than exposure to single bacteria. Donor 2 also presented with similar pattern but with lower IL-10 levels. These results suggested that the anti-inflammatory response was triggered when BV bacterial extracts were combined with those from Lactobacillus strains similar to the THP-1 cell lines (Figure 8.0). 3C.6.2 Pro-inflammatory cytokines 3C.6.2.1 IL-1β produced by MD-DCs Figure 13.0 presents the IL-1β produced by the MD-DCs from two donors. Donor 3 MD-DCs produced more IL-1β than Donor 2. Exposure to single bacteria triggered less IL-1β than combination of lactobacilli and a single BV organism. Combination of L. gasseri + G. vaginalis triggered highest IL-1β than L. reuteri + G. vaginalis. Interestingly the negative control for donor 3 appeared to trigger a much higher level of IL-1β than the negative control for donor 2. This could be due to differences in the health of each individual at the time the blood sample was taken. 3C.6.2.2 IL-6 production by MD-DCs Figure 14.0 compares IL-6 levels between MD-DCs from two donors. Donor 3 responded with increased levels of IL-6 in response to combination of bacteria: L. 66 gasseri + A. vaginae (9362.5 pg/ml), L. reuteri + A. vaginae (7681 pg/ml) and L. reuteri + G. vaginalis (3187 pg/ml) with the least by L. gasseri + G. vaginalis (2675 pg/ml). The single bacteria indicated L. gasseri triggered less IL-6 response than the others for donor 3 whilst G. vaginalis triggered the least IL-6 for Donor 2. The combination of L. gasseri + A. vaginae tended towards a synergistic effect whilst an additive effect appears to be occurring with L. reuteri + G. vaginalis. IL-6 can be associated with the generation of predominantly antibody-mediated TH2 response so in this instance, may represent an anti-inflammatory response. 3C.6.2.3 IL-8 production by MD-DCs In figure 15.0 IL-8 produced by the MD-DCs also indicated increased levels ranging from 21880 pg/ml (G. vaginalis) to 28427 pg/ml (L. gasseri + A. vaginae) for donor 3. For donor 2, the IL-8 levels ranged from 22023 pg/ml (G. vaginalis) to 28752 pg/ml (L. gasseri + G. vaginalis) and the negative control with 16009 pg/ml. These high results indicated the sensitivity of bioplex assay system as compare to ELISA and a dilution of supernatants would be again necessary if the exact values are to be determined. 3C.6.2.4 IL-12 production by MD-DCs From the results in figure 16.0 shows IL-12 produced from MD-DCs from donor 2 ranged from 0 pg/ml (negative control) to 935 pg/ml (L. reuteri) and Donor 3 has IL-12 levels ranging from 0 pg/ml (L. gasseri) to 249 pg/ml (L. reuteri). These data suggest that BV bacterial extracts may actually suppress IL-12 production in response to L . reuterii 3C.6.2.5 TNF-α production by MD-DCs As shown in Figure 17.0 presents the TNF-α level for donor 2 ranged from 3746.5 pg/ml (negative control) to 25912 pg/ml (L. gasseri + A. vaginae) and donor 3 with values ranging from 1357.5 pg/ml (negative control) to 26870.5 pg/ml (L. gasseri + A. vaginae). The combination of lactobacilli with one of the BV organisms yielded more TNF-α than single organism reflecting the results using the ELISA assay but with higher sensitivity of bioplex. 67 TGF‐beta (pg/ml) 700 600 500 400 300 200 100 0 Donor 2 Donor 3 Figure 11.0 Bioplex assay for TGF-beta levels by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts. 68 3000 2500 IL‐10 (pg/ml) 2000 1500 D2 1000 D3 500 0 Figure 12.0 Bioplex assay for IL-10 levels by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts. 69 300 250 IL‐1B (pg/ml) 200 150 Donor 2 100 Donor 3 50 0 Figure 13.0 Bioplex assay for IL-1ß levels by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts. 70 10000 9000 8000 IL‐6 (pg/ml) 7000 6000 5000 4000 D2 3000 D3 2000 1000 0 Figure 14.0 Bioplex assay for IL-6 levels by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts. 71 35000 30000 IL‐8 (pg/ml) 25000 20000 15000 D2 10000 D3 5000 0 Figure 15.0 Bioplex assay for IL-8 levels by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts. 72 1000 900 800 IL‐12 (pg/ml) 700 600 500 400 300 Donor 2 Donor 3 200 100 0 Figure 16.0 Bioplex assay for IL-12 levels by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts 73 30000 TNF alpha in pg/ml 25000 20000 15000 Donor 2 10000 Donor 3 5000 0 Figure 17.0 Bioplex assay for TNF-α levels produced by MD-DCs from two donors in response to lactobacilli and BV bacterial extracts 74 3D Discussion 3D.1 THP-1 cells, MD-DCs and MD-LCs The monocytes, macrophages and dendritic cells are the primary effector immune cells of the innate immune system. They have an important role as APCs and further regulate the balance of cytokines and chemokines in the adaptive immune system (Vaarala, 2003) and recent work has shown that Lactobacillus strains induce and modulate the cytokine profile from monocytic derived macrophage and dendritic cells (Livingston et al., 2010), (Christensen et al., 2002). The THP-1 immortalised cell line has provided limitless supplies of monocytic cells which are easily differentiated with PMA to generate macrophages. The availability of macrophage has enable immunomodulatory studies of cytokines of interest in this study. An interesting observation is that during differentiation process, the proliferation rate of the immortalised cells is halted and within 48-72 hr the monocytic cells are fully differentiated into macrophages (Schwende et al., 1996). The methods being used to generate dendritic cells from human blood has offered limited supplies of immune cells as compared to the THP-1 cells. The yield of MDDCs obtained per donor varied and hence tests done were not able to be repeated nor carried out in triplicate. Furthermore, not all lactobacilli and controls were tested for because of inadequate quantity yield. However this study has shed some light into providing preliminary tests and results which could pave the way for further thorough investigation on the immunomodulatory effects of potential probiotics against the BV bacteria. 3D.2 Optimisation assays & preliminary testing Initially it was important to optimise the concentration of THP-1 cells needed to completely differentiate, the dose of differentiation agent (PMA in this case) and the bacterial cell concentration that would trigger measureable cytokines. The inclusion of LTA and LPS as positive controls represented the effects of Gram positive and Gram negative bacteria on the immune cells in vivo during BV. 75 3D.2.1 Optimising PMA and THP-1 cell concentration Exposing 2.5x106 cells/ml THP-1 cells to 20 ng/ml of PMA for 48 hr was found to induce adequately differentiation of monocytes to macrophage in this study. This was established by using both 1% crystal violet staining to detect the adherence of the differentiated cells and trypan blue exclusion to detect viable/non-viable cells. The degree of differentiation was further confirmed by testing the amount of TNF-α produced in response to exposing the differentiated THP-1 cells to LPS, LTA and the bacterial extracts of lactobacilli and BV bacteria. A number of studies have demonstrated that the minimum cell concentration of THP-1 cells needed to differentiate from monocytes to macrophage and the dose of PMA required have varied. Schwende et al used 2.5x105 cells/ml with 10-8 M of PMA for 48 hours to obtain differentiated macrophages that responded to LPS with enhanced TNFα production (Schwende et al., 1996). Another study indicated that 5 ng/ml of PMA was sufficient to trigger THP-1 cells (1x106 cells/ml) to fully differentiated after 48 hr without undesirable gene up-regulation when exposed to 10 ng/ml of LPS (Park et al., 2007). Interestingly a study by Daigneault et al used 2 x 105 cells/ml THP-1 cells differentiated with 200 nM PMA for 3 days followed by further incubation of the cells with RPMI 1640 medium plus 10 % FCS to day 5 demonstrated macrophages closely resemble the human monocytic derived macrophage (Daigneault et al., 2010). The results found in our study are within this range thus demonstrating consistency with other findings reported in the literature. 3D.2.2 Optimising bacterial load, LPS and LTA to trigger TNF-α response From the optimisation stage the bacterial load of 5x108 cfu/ml was shown to trigger more TNF-α as compared to lower concentrations of bacteria tested. However, the G. vaginalis did not respond in a similarly trend to other bacteria tested. The possible reasons for this could be pipetting errors or bacterial deposits inadvertently washed out when supernatants were removed after centrifugation. Numerous studies carried out to investigate cytokine induction by lactobacilli had demonstrated previously that a higher bacterial load was required to stimulate cytokine production by immune cells. A range of 106 to 109 cfu/ml lactobacilli was used to 76 stimulate cytokines from dendritic cells, 106 and 107 cfu/ml of Lactobacillus strains to induce cytokines from human peripheral blood mononuclear cells while 1x103 cfu of lactobacilli per single human myeloid dendritic cell (Hart et al., 2004), (Vissers et al., 2010), (Mohamadzadeh et al., 2005). Throughout these experiments LPS and LTA were used as controls for the Gram negative and Gram positive bacteria, as mentioned previously. LPS or endotoxin is a major component of the Gram negative bacterial cell wall. It is a strong activator of the immune cells including macrophages, monocytes and endothelial cells inducing production of proinflammatory cytokines (Zhang et al., 1999). On the other hand, LTA is a component of the Gram positive bacterial cell wall which represents the composition of Lactobacillus cell wall. The dose used in the experiments described here was derived from the literature and confirmed in current work in the laboratory 3D.3 TNF-α production by THP-1 cells in response to Lactobacillus strains and BV bacteria measured by ELISA The proinflammatory cytokines TNF-α, IL-1β and IL-6 are usually the first cytokines induced in response to pathogenic bacteria products (Gabay, 2006). In this regard, the TNF-α measurement was initiated to investigate whether Lactobacillus strains used were able to induce TNF-α. An ELISA was used as an initial assay followed by the much more sensitive Bioplex multiplex assay in which a range of other proinflammatory and suppressive cytokines potentially produced by both THP-1 and MD-DCs used in this study, was assessed. There were various amounts of TNF-α produced by the THP-1 cells after exposure to the six Lactobacillus strains. This indicated that proinflammatory cytokine production was triggered in the immune cells in response to some of the Lactobacillus strains. In support of these results, other studies have also indicated that Lactobacillus strains are commonly strong inducers of TNF-α cytokine (Miettinen et al., 1996), (Klebanoff et al., 2004),(Vissers et al., 2010). However, L. rhamnosus induced the least TNF-α out of all the Lactobacillus strains. This reflects a similar finding from a study in which the L. rhamnosus GG and GR-1 strains were found to be not strong TNF-α inducers (Kim et al., 2006a).The presence of the LTA which is a component of the Gram positive bacteria is proposed to be the main stimulator of TNF-α through TLR 2 (Matsuguchi et 77 al., 2003). The immunomodulating properties of each lactobacilli are strain specific and hence each specific strain must not be used in place to other strains in the probiotic context. Furthermore, the expected marked inhibitory action of the lactobacilli upon the BV bacteria cytokine production as hypothesised was not found for all of the lactobacilli strain when each was combined with the BV bacteria. This could be because these Lactobacillus strains are strong TNF-α inducers themselves which may be attributable to the LTA composition of each strain. As LPS is also a molecule shown to elicit TNFα it could be present in the cell wall composition of the BV bacteria despite being Gram variable. Thus the combination of lactobacilli and BV bacteria would induce increased amount of TNF-α from the THP-1 cells additively. 3D.4 TNF-α production by MD-DCs from 3 female donors in response to Lactobacillus strains and BV bacteria measured by ELISA To test the MD-DCs TNF-α response to lactobacilli and BV bacteria, only 2 Lactobacillus strains were used. L. reuteri was included because of its known background as a probiotic whilst L. gasseri was chosen on the basis of it being a dominant Lactobacillus strain in the normal flora of the vagina. The quantity of TNF-α produced by the MD-DCs in response to the Lactobacillus strains and BV bacteria varied between the 3 female donors. However, there was an obvious trend towards a synergistic effect when L. gasseri was combined with BV bacteria observed. The desired probiotic inhibitory effect upon BV bacteria was not demonstrated by the 2 potential Lactobacillus strains tested as per hypothesised. The difference in immune responses to the bacterial challenges seen from the 3 donors could be mainly due to the different genetic makeup of each individual. Moreover, testing of all the Lactobacillus strains was impossible at this point in time due to the limited quantity of MD-DCs obtained per donor. This has also prevented the tests to be done in triplicate to achieve a much more reproducible result per donor therefore such are taken as preliminary results only. Further work is therefore needed to 78 investigate the possible inhibitory effects of other Lactobacillus strains upon BV bacteria on the MD-DCs immune cells. The use of ELISA was a preliminary test for TNF-α production by THP-1 cells and MD-DCs in response to exposure to the lactobacillus strains and BV bacteria. Further testing by Bioplex assay gave an in-depth of cytokine response profile as discussed below. 3D.5 Anti-inflammatory cytokines produced by THP-1 cells in response to Lactobacillus strains and BV bacteria measured by Bioplex assay The anti-inflammatory response of the immune cells was further investigated using the Bioplex assay as mentioned earlier. The immunosuppressive cytokines produced by the THP-1 cells in response to lactobacilli are of interest because the production of these may alter the balance between the pro-inflammatory and anti-inflammatory cytokines favouring a down regulatory environment. The TGF-β1 is a protein molecule that controls proliferation and cellular differentiation. Its involvement in immune system is to regulate the development of regulatory T cells and also to block activation of lymphocytes and monocyte- derived phagocytes and downregulation of pro-inflammatory cytokines TNF-α, IL-6 and IL-12 (Chen et al., 2005). Interestingly, we observed high production of TGF-β1 in THP1 cells alone i.e. the negative control which may be due to its other role in the cell cycle controlling cellular proliferation and differentiation. The THP-1 cells are immortalised and when used in these experiments were differentiated into macrophages. The detection of this cytokine may then be attributable to the nature of the cell type. The increased production of IL-10 as seen when the L. gasseri and L. rhamnosus were in combination with the BV bacteria indicated that lactobacilli studied have the potential to skew towards anti-inflammatory responses. This was a very interesting and potentially important result. In support of this, a previous report has shown that IL-10 79 was induced by other Lactobacillus strains upon exposure to human PBMC (Vissers et al., 2010). Interestingly, a study by Kim et al has postulated that the inducement of IL10 by L. rhamnosus GG and GR-1 did not directly suppress TNF-α production, but rather it was through inducement of the granulocyte-colony stimulating factor (G-CSF). Further, THP-1 cells have high affinity G-CSF receptors which have been shown to favour this pathway (Kim et al., 2006b). 3D.6 Pro-inflammatory cytokines produced by THP-1 cells in response Lactobacillus strains and BV bacteria measured by Bioplex assay The inducement of IL-1β by all the Lactobacillus strains and BV bacteria both singly and in combination demonstrated the capability of these bacteria to induce proinflammatory response in the THP-1 cells. IL-1β is among the earliest pro- inflammatory cytokines to be produced in response to infection and its production signifies it is induced by all bacteria whether they be probiotic or pathogenic. Vissers et al also showed that the Lactobacillus strains studied induced IL-1β, further supported by Miettinen et al (Vissers et al., 2010), (Miettinen et al., 1998). IL-6, one of the earlier pro-inflammatory cytokines, was produced by THP-1 cells in response to exposure to all the Lactobacillus strains and BV bacteria. In support of the findings 2 previous studies have verified their production by immune cells (Vissers et al., 2010), (Miettinen et al., 1998). However as the IL-6 values for all the bacteria tested were in excess of 25000 pg/ml, the assay needs to be carried out again with the supernatants diluted in order to gain accurate results; this could probably yield very high amounts and may discriminate between different bacterial stimuli. IL-1β produced by mononuclear phagocytes is known to activate macrophages to produce IL-6 (Dinarello, 1996). Furthermore, IL-6 is a pleitropic cytokine which can act as both an anti-inflammatory and pro-inflammatory cytokine. It is produced by monocytes/macrophages in response to bacterial peptides and endotoxin (Papanicolau et al., 1998). I would speculate here that the increased production of could be due to both the exposure to bacterial cell from the lactobacilli and BV bacteria as well as the effect of IL-1β. 80 The IL-8 cytokine measurement also indicated high levels including the negative control. The most likely reason would be some contamination of the negative control with bacterial cells from the lactobacilli and BV bacteria. However, its production in response to the bacterial cells indicated this chemokine is also induced. Its main function as a powerful chemoattractant to neutrophils and promotion of the cell mediated immune response (Boyle, 2005). Unfortunately, the production of IL-12 by THP-1 cells in response to the lactobacilli and BV bacteria was too low to be functionally significant. Nonetheless, this TH1 type cytokine did appear to show some strain specificity so it is conceivable that some Lactobacillus strains may induce it whilst others may not. In support of this, another study has shown that IL-12 was strongly induced by L. rhamnosus E509 (Miettinen et al., 1998). The greatly improved sensitivity of the Bioplex assay was demonstrated when TNF- α was quantitated in culture supernatants and compared to the data obtained using an ELISA. Again, to discern the differences, titration is required however with time constraint this was not possible. But nevertheless, the production of TNF-α in response to both the Lactobacillus strains and BV bacteria indicated that it is also one of the earlier cytokines to be produced in high quantities in response to bacterial exposure regardless of the type of bacteria. 3D.7 Anti-inflammatory cytokines produced by MD-DCs in response to Lactobacillus strains and BV bacteria measured by Bioplex assay The 2 anti-inflammatory cytokines assayed in THP-1 supernatants were also assayed in MD-DCs culture supernatants. The production of TGF-β by the negative control could be due to the same reason as stated for THP-1 cells. The production of IL-10 by MD-DCs however indicated an anti-inflammatory response similar to the THP-1 cells. It has also been found from previous studies that Lactobacillus strains augmented the production of IL-10 to different degrees. L. reuteri DSM 12246 strongly induced IL-10 than L. rhamnosus GG (Zeuthen et al., 2006) whilst VSL#3 induced IL-10 drives the inflammatory towards a regulatory profile (Hart 81 et al., 2004). The reproducibility of this result in an immortalised cell line and in cells derived from a primary source is encouraging and deserves further investigation. 3D.8 Pro-inflammatory cytokines produced by MD-DCs in response Lactobacillus strains and BV bacteria measured by Bioplex assay IL-1β produced by MD-DCs from the 2 donors also demonstrated differences in the individual immune response to the Lactobacillus strains and BV bacteria. MD-DCs from one donor produced very little IL-1β but supernatant from the cells from the other suggested that L. reuteri may have some suppressive potential on one of the BV strains, G. vaginalis. It would therefore be worth investigating this further using a larger cohort of donors. 3D.9 Conclusion and future directions As such results obtained from this study are just preliminary, further work on the other Lactobacillus strains is crucial. There are possibilities that the other strains might upregulate the anti-inflammatory cytokines which would drive the immune responses of the host (pregnant women with BV) to prevent spontaneous abortions or preterm labour. It should be emphasised that altering the balance of proinflammatory and anti inflammatory cytokines towards a suppressive millieu rather than an ‘all-or-nothing’ effect may be very important in defining the efficacy of a given strain of lactobacilli as a probiotic for pregnant women . 82 References 1. 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Microbiol. 150, 2565-2573. 100 APPENDICES Appendix A The standard curves of Lactobacillus strains A.1 Standard curve of L. crispatus 1.00E+08 9.00E+07 8.00E+07 cfu/ml 7.00E+07 6.00E+07 L.crispatus Cfu/ml 5.00E+07 4.00E+07 Linear(L.crispatus Cfu/ ml) 3.00E+07 2.00E+07 1.00E+07 0.00E+00 0 0.2 0.4 0.6 0.8 Absorbance A.2 Standard curve of L. acidophilus 1.40E+08 1.20E+08 1.00E+08 cfu/ml 8.00E+07 L.acidophilus Cfu/ml 6.00E+07 Linear(L.acidophilus Cfu/ ml) 4.00E+07 2.00E+07 0.00E+00 0 ‐2.00E+07 0.2 0.4 0.6 0.8 1 Absorbance 101 A.3 Standard curve of L. paracasei 1.60E+08 1.40E+08 1.20E+08 cfu/ml 1.00E+08 8.00E+07 L.paracasei Cfu/ ml 6.00E+07 4.00E+07 2.00E+07 0.00E+00 ‐2.00E+07 0 0.2 0.4 0.6 0.8 Absorbance A.4 Standard curve of L. rhamnosus 8.00E+08 7.00E+08 6.00E+08 cfu/ml 5.00E+08 L.rhamnosus cfu 4.00E+08 Linear(L.rhamnosus cfu) 3.00E+08 2.00E+08 1.00E+08 0.00E+00 ‐1.00E+08 0 0.2 0.4 0.6 0.8 1 Absorbance 102 A.5 Standard curve of L. reuteri 1.40E+08 1.20E+08 1.00E+08 cfu/ml 8.00E+07 6.00E+07 L.reuteri cfu/ml 4.00E+07 Linear(L.reuteri cfu/ml) 2.00E+07 0.00E+00 ‐2.00E+07 0 0.2 0.4 0.6 0.8 1 Absorbance A.6 Standard curve of L. gasseri 3.00E+08 2.50E+08 cfu/ml 2.00E+08 1.50E+08 L.gasseri cfu/ml 1.00E+08 Linear(L.gasseri cfu/ml) 5.00E+07 0.00E+00 0 ‐5.00E+07 0.2 0.4 0.6 0.8 1 Absorbance 103 A.7 Standard curve of G. vaginalis 6.00E+08 5.00E+08 cfu/ml 4.00E+08 3.00E+08 G.vaginalis cfu 2.00E+08 1.00E+08 0.00E+00 0 0.02 0.04 0.06 Absorbance 0.08 0.1 A.8 Standard curve of A. vaginae 4.00E+07 3.50E+07 3.00E+07 cfu/ml 2.50E+07 2.00E+07 A.vaginae cfu 1.50E+07 Linear(A.vaginae cfu) 1.00E+07 5.00E+06 0.00E+00 0 0.02 0.04 0.06 0.08 Absorbance 104 Appendix B TMB-Plus culture media recipe Component Amount Brucella agar (BD) 43 g Distilled water 1000 ml TMB (Sigma) 250 mg Starch solution (BD) 20 g Hemin Solution (Sigma) 10 ml Vitamin K1 solution (Sigma) 0.2 ml Magnesium sulphate, anhydrous 0.57 g Magnesium sulphate monohydrate 0.12 g Peroxidase solution (1mg/ml) 10 ml (horse radish peroxidise,Sigma) Horse serum (Gibco/Life Technologies) 50 ml Agar(BD) 10 g 105 Appendix C ELISA REAGENTS Phosphate coating buffer for TNF-α 0.1M Na2 HPO4 adjusted to pH 9.0 with 0.1M NaH2PO4 Blocker 1x PBS + 1% Bovine serum albumin (BSA) 1% BSA= 1 g BSA + 100 ml of PBS Wash buffer PBS + 0.05% Tween 20= 2L 1x PBS + 1 ml Tween 20 4 N H2SO4 54 ml of H2SO4 (18.4M concentration) to 446 ml distilled water (Stock) Dilute 1 in 4 parts distilled water to obtain 1N H2SO4 106 Appendix D 35000 30000 Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 25000 Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 20000 L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag 15000 L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag 10000 L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control 5000 0 29500 29000 L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control Bio-Plex results IL‐6 (pg/ml) 28500 28000 27500 27000 26500 26000 Bioplex assay for IL-6 and IL-8 levels by THP-1 cells in response to lactobacilli and BV bacterial extracts. IL‐8 (pg/ml) Expt 1 Expt 2 Expt 3 Expt 1 Expt 2 Expt 3 107 Lacido+Avag Lreut+Avag Lcrisp+Avag L para+Avag Lgas+Avag Lrham+Avag 30000 L.acido+Gvag L.reut+G vag Lcrisp+Gvag L.para+Gvag Lgas+Gvag Lrham+Gvag 25000 L.acido L.reut L.crisp L.para L.gas L.rham G vag A vag LPS LTA Neg control 20000 15000 10000 5000 0 Bioplex assay for TNF-α level by THP-1 cells in response to lactobacilli and BV bacterial extracts TNF‐alpha (pg/ml) 108 Expt 1 Expt 2 Expt 3