THFS‘IS “IV " /: L1») 1. LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. ,4 T DATE DUEA ' DATE DUE W ,e m 0124294 384842110 0" E LL- O N» i454 {p0 6’01 c:/ClFiC/DatoDuo.p65-p. 15 EFFECT OF LACTIC ACID BACTERIA AND BIFIDOBACTERIA ON INTERLEUKIN-6 AND lNTERLEUKlN-8 PRODUCTION BY CACO-2 CELLS By Constance Wong A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 2002 UMI Number: 1409566 ® UMI UMI Microform 1409566 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT EFFECT OF LACTIC ACID BACTERIA AND BIFIDOBACTERIA ON INTERLEUKIN-6 AND INTERLEUKlN-S PRODUCTION BY CACO-Z CELLS By Constance Wong The purpose of this study was to test the hypothesis that probiotics could enhance immune function by stimulating cytokine secretion by intestinal epithelial cells. To test this hypothesis, the effects of fermented and non-fermented reconstituted non-fat dry milk containing probiotic cultures (Lactobacillus acidophilus, L. bulgaricus, L. casei, L. reuteri, Streptococcus thermophilus, Bifidobacterium, B. adolescentis) on interleukin (IL)-6 and IL-8 production by Caco-2 cells were assessed. Three different concentrations (106, 107, 108 CFU/ml) of probiotic cultures were used to determine the optimum dose to elicit a maximal immune response. Probiotic cultures were inactivated by heat (95°C, 30 min) or irradiation (l Mrad). In addition, milk components (lactose, a-lactalbumin, B- lactoglobulin) were evaluated for their ability to stimulate IL-6 and IL-8 production. In general, none of the cultures investigated significantly stimulated IL-6 or IL-8 production. There was a significant difference, however, between heat- and irradiation- inactivated samples. Heat-inactivated cultures caused more IL-6 and IL-8 production than their irradiated counterparts. These results suggest that the mode of inactivation may be important to immune stimulation. The milk components, a-lactalbumin and B- lactoglobulin elicited markedly high amounts of IL-6 and IL-8 production from Caco-2 cells. These results suggest that certain milk components have immunostimulating abilities in the gastrointestinal tract. ACKNOWLEDGMENTS I would like to thank Dr. Zeynep Ustunol, my major professor, for her guidance and assistance throughout my time at Michigan State. Thanks are also given to Dr. James Pestka for his expertise and accessibility whenever I needed it, and Dr. Elliot Ryser for his support as a committee member during my research. Shama Joseph, Emily Smith and Matthew Rarick played important roles in various stages of my research. I could not have made it this far without the support of my friends and family. iii TABLE OF CONTENTS vi LIST OF TABLES LIST OF FIGURES viii ABBREVIATIONS x Chapter Page 1 INTRODUCTION 1 2 LITERATURE REVIEW 5 2.1 Intestinal microflora 6 2.1.1 Lactic acid bacteria and bifidobacteria 7 2.1.2 Probiotics 10 2.2 GI immune system 12 2.2.1 Cytokines 14 2.2.2 Effect of probiotics on immune 15 responses in the GI tract 2.3 Immunostimulating effects of lactic acid bacteria 17 bifidobacteria, and milk components 2.3.1 In vitro studies 17 2.3.2 Animal studies 21 2.3.3 Human/clinical studies 26 2.3.4 Effect of milk components 29 2.4 Caco-2 cells 34 2.5 Rationale for this research 39 3 MATERIALS AND METHODS 40 3.1 Culture preparation 41 3.2 Caco-2 cell culture 45 3.3 Stimulation of cytokine production by probiotic 46 bacteria 3.4 IL-6 and IL-8 quantitation 46 3.5 Stimulation of cytokine production of Caco-2 cells by milk 47 components 3.6 Statistical analysis 48 4 RESULTS AND DISCUSSION 49 4.1 Effect of lactic acid bacteria and bifidobacteria on IL-6 50 production by Caco-2 cells iv 4.1.1 Effect of culture 4.1.2 Effect of dose 4.1.3 Effect of fermentation 4.1.4 Effect of inactivation 4.1.5 Effect of the interaction between culture and dose 4.1.6 Effect of the interaction between culture and inactivation 4.1.7 Effect of the interaction between culture and fermentation 4.1.8 Discussion on the effect of lactic acid bacteria and bif'rdobacteria on IL-6 production by Caco-2 cells 4.2 Effect of lactic acid bacteria and bifrdobacteria on IL-8 production by Caco—2 cells 4.3 Future research for lactic acid bacteria and bifrdobacteria 4.2.1 Effect of culture 4.2.2 Effect ofdose 4.2.3 Effect of fermentation 4.2.4 Effect of inactivation 4.2.5 Effect of the interaction between culture and dose 4.2.6 Effect of the interaction between culture and inactivation 4.2.7 Effect of the interaction between culture and fermentation 4.2.8 Effect of the interaction between ferment- tation and concentration 4.2.9 Discussion on the effect of lactic acid bacteria and bif'rdobacteria on IL-6 production by Caco-2 cells in NFDM on cytokine production by Caco-2 cells 4.4 Effect of milk components on cytokine production by Caco-cells 5 SUNIMARY LIST OF REFERENCES APPENDIX I APPENDR II 4.4.] Future research 5] 51 57 57 58 58 63 63 66 67 72 72 72 73 76 79 81 84 84 89 9O 92 103 105 LIST OF TABLES Table 2.1 Recent in vitro studies of immune stimulation by lactic acid bacteria Table 2.2 Recent animal studies of immune stimulation by lactic acid bacteria Table 2.3 Recent human studies of immune stimulation by lactic acid bacteria Table 2.4 Recent studies of immunostimulation by milk components Table 2.5 Immune effects of bioactive milk peptides Table 2.6 Studies on stimulation of cytokine production by Caco-2 cells Table 3.1 Sources of bifrdobacteria and lactic acid bacteria used in this study Table 4.] ANOVA table comparing non-fermented cultures at concentrations of 106, 107, and 108 CFU/ml on IL-6 production by Caco-2 cells after 24 h incubation Table 4.2 ANOVA table comparing non-fermented cultures at concentrations of 106, 107, and 108 CFU/ml on IL-6 production by Caco-2 cells after 48 h incubation Table 4.3 ANOVA table comparing fermented and non-fermented cultures at concentrations of 106 and 107CFU/ml on H36 production by Caco-2 cells after 24 h incubation Table 4.4 ANOVA table comparing fermented and non-fermented cultures at concentrations of 106 and 107 CFU/ml on IL-6 production by Caco-2 cells after 24 h incubation Table 4.5 ANOVA table comparing non-fermented cultures at concentrations of 106, 107, and 108 CPU/ml on lL-8 production by Caco-2 cells after 24 h incubation Table 4.6 AN OVA table comparing non-fermented cultures at concentrations of 106, 107, and 108 CFU/ml on IL-8 production by Caco-2 cells after 48 h incubation Table 4.7 ANOVA table comparing fermented and non-fermented cultures at concentrations of 106 and 107CFU/ml on IL-8 production by Caco-2 cells after 24 h incubation vi 18 22 27 3O 31 36 42 52 53 55 56 68 69 70 Table 4.8 ANOVA table comparing fermented and non-fermented cultures at 71 concentrations of 106 and 107 CFU/ml on IL-8 production by Caco-2 cells after 24 h incubation vii LIST OF FIGURES Figure 2.1 Changes in human fecal flora as age increases 9 Figure 2.2 The gut-associated lymphoid tissue (GALT) 13 Figure 2.3 Transwell cell culture system 33 Figure 3.1 Schematic diagram of probiotic sample preparation 44 Figure 4.1 Effect of non-fermented probiotic cultures at concentrations of 106, 54 107, and 108 CFU/ml on IL-6 production by Caco-2 cells (48 h incubation) Figure 4.2 Effect of heat and irradiation inactivation of non-fermented lactic acid 59 bacteria and bifidobacteria at concentrations of 106, 107, and 108 CFU/ml on IL-6 production by Caco-2 cells (24 h incubation) Figure 4.3 Effect of heat and irradiation inactivation of non-fermented lactic acid 61 bacteria and bifrdobacteria at concentrations of 106, 107, and 108 CFU/ml on IL-6 production by Caco-2 cells (48 h incubation) Figure 4.4 Effect of heat and irradiation inactivation of fermented and non- 62 fermented lactic acid bacteria and bifrdobacteria at concentrations of 106 and 107 CFU/ml on IL-6 production by Caco-2 cells (24 h incubation) Figure 4.5 Comparison of non-fermented, heat- and irradiation-inactivated LB on 74 IL-8 production by Caco—2 cells (48 h incubation) Figure 4.6 Comparison of lactic acid bacteria and bifidobacteria at concentrations 75 of 106 and 107 CFU/ml on IL-8 production by Caco-2 cells (48 h incubation) Figure 4.7 Effect of heat and irradiation inactivation of non-fermented lactic acid 77 bacteria and bifrdobacteria at concentrations of 106, 107, and 108 CFU/ml on IL-8 production by Caco-2 cells (48 h incubation) Figure 4.8 Effect of heat and irradiation inactivation of fermented and non- 78 fermented lactic acid bacteria and bifidobacteria at concentrations of 106 and 107 CFU/ml on IL-8 production by Caco-2 cells (24 h incubation) Figure 4.9 Effect of heat and irradiation inactivation of fermented and non- 79 fermented lactic acid bacteria and bifidobacteria at concentrations of 106 and 107 CFU/ml on IL-8 production by Caco-2 cells (48 h incubation) Figure 4.10 Effect of various milk components on IL-6 production by Caco-2 cells 85 with or without stimulation by IL-1 B viii Figure 4.11 Effect of various milk components on IL-8 production by Caco-2 cells 87 with or without stimulation by IL-1 [3 ix AIDS ANOVA or-la ATCC B-Ig BSA BGG CFU CN ConA DMEM ELISA ETEC FBS GALT GI GM-C SF IEL ABBREVIATIONS Acquired immunodeficiency syndrome Analysis of variance or-Lactalbumin American Type Culture Collection B-Lactoglobulin Bovine serum albumin Bovine gamma globulin Colony forming unit Casein Concanavalin A Dulbecco’s modified Eagle’s media Enzyme-linked immunosorbent assay Enterotoxigenic Escherichia coli Fermented Fetal bovine serum Gut-associated lymphoid tissue Gastrointestinal Granulocyte-macrophage colony-stimulating factor Heat-killed cells Irradiated Interepithelial lymphocyte ‘ Interferon LPS MCP- 1 MRSL NFDM NO 0A OD OM PBMC PBS PBST PMN PP TGF-B TLR TNF-a Immunoglobulin Interleukin Lactic acid bacteria Lipopolysaccharide Monocyte chemotactic protein-1 De Man, Rogosa, Sharpe medium for lactobacilli De Man, Rogosa, Sharpe medium with 5% lactose Non-fermented Non-fat dry milk Natural killer Nitric oxide National Yogurt Association Ovalbumin Optical density Ovomucoid Peripheral blood mononuclear cell Phosphate buffered saline Phosphate buffered saline with 0.05% Tween-20 Polymorphonuclear Peyer’ 3 Patch Transforming growth factor beta Toll-like receptor Tumor necrosis factor alpha xi CHAPTER 1 INTRODUCTION CHAPTER 1 INTRODUCTION The relationship between food, nutrition and health has long been known to exist. While early research focused on foods to prevent disease, current research is focused on foods to prolong and enhance health. Functional foods are generally defined as foods that provide a health benefit beyond inherent nutrition. This category of foods includes infant formulas, medical foods, dietary supplements, performance foods, probiotics and other foods designed to deliver specific nutrients or food components (i.e. fiber, vitamins, minerals, antioxidants, probiotics, dairy proteins, soy proteins and lipids). Dairy products have been the focus of functional food research due to the bioactive properties of some milk components and their ability to serve as excellent carriers for probiotic organisms. Because of the perceived health benefits of probiotics, consumption and sales of yogurt in particular, rose to $2.2 million in 2001, which was a 6.6% increase from 2000 (Berry, 2002). This trend has been attributed to greater consumer interest in nutrition and health as well as the increase in published studies that indicate yogurt cultures may have additional health benefits. The consumption of fermented milks has been associated with improved health for thousands of years. Hippocrates (circa 400 BC), the father of medicine, considered fermented milks to have medicinal qualities and prescribed them for stomach and intestinal ailments (Oberman, 1985). Eli Metchnikotf (1907) was the first to document the improved health of patients ingesting milk fermented by lactic cultures. According to his speculations, those who ingested fermented milk lived longer because the bacteria in the milk helped to maintain a healthier intestine by decreasing toxic microbial activities. These health-promoting bacteria were identified as probiotic lactic acid bacteria (LAB). Since Metchnikoff, many reports have described the benefits probiotics have on human health. Among them are the alleviation of lactose intolerance symptoms and diarrhea, anti-cancer effects, reduced serum cholesterol, and enhanced immune response (Fuller, 1991; Gilliland, 1990). Probiotics are thought to exert immune effects via the gastrointestinal (GI) tract where they interact with the gut-associated lymphoid tissue (GALT). It is believed that probiotic interaction leads various immune cells in the GI tract to mount an immunological response. Several studies have demonstrated the ability of probiotics to enhance both non- specific and specific immune responses in humans (Gill and others, 2001; Donnet-Huges and others, 1999; Schiffiin and others, 1995). The efficacy of probiotics in humans was based on levels of immunoglobulins (1g) and immune cells in blood rather than on stimulation of cytokines. An increase in Ig indicates that the body’s adaptive immunity is responding to infection by a foreign substance (antigen). An increase in cytokine production, however, occurs via the innate immune response to recruit more phagocytic cells and effector molecules to the site of infection (Janeway and others, 1999). Most in vitro studies looked at cyokines but used mouse rather than human cell lines. We have chosen the Caco-2 cell line, which is considered a good model for human intestinal epithelial cells, in order to address issues concerning cytokine stimulation of cells by probiotic bacteria. The hypothesis on which this research was based was that LAB and bifidobacteria, which are used in the production of fermented dairy foods, could enhance immune function by stimulating cytokine secretion by intestinal epithelial cells. Therefore, the objectives of this research were as follows: 1) Examine the difference between fermented and non-fermented non-fat dry milk (NFDM) containing seven individual probiotic cultures on cytokine production by Caco-2 cells 2) Determine optimum levels of probiotic organisms to elicit a maximal immune response by Caco-2 cells 3) Investigate the effect of heat or irradiation inactivated cells on the stimulation of cytokine secretion by Caco-2 cells 4) Examine the effect of specific milk components on cytokine production by Caco-2 cells CHAPTER 2 LITERATURE REVIEW CHAPTER 2 LITERATURE REVIEW 2.1 Intestinal microflora More than 400 species of bacteria are thought to inhabit the large intestine (Finegold and others, 1983) and make up to 40-55% of fecal solids for those on a western-type diet (Cabotaje and others, 1990). The dominant group of microflora is obligately anaerobic and includes the bacteria bacteroides, eubacteria, bifidobacteria, lactobacilli, anaerobic cocci and clostridia (Kleessen and others, 2000). Naturally occurring microfiora serve as a protective barrier against invasion by pathogens (Tancrede, 1992). Most of these bacteria are found in the large intestine as the constant flow of gut contents keeps numbers in the small intestine relatively low (Pestka, 1993). Commensal bacteria offer protection to the host from pathogens by blocking or attaching to receptors, competing for nutrients and by producing antimicrobial compounds (Vaughan and others, 1999). Though naturally occurring microflora exert these beneficial effects, their main role is to ferment carbohydrates (not digested earlier in the gut) to provide additional energy (Cummings and Macfarlane, 1991). The composition of commensal microflora varies between individuals, but the population is fairly stable in healthy adults (Kleessen and others, 2000). When the balance of microflora is disturbed due to advanced age, diet, illness or antibiotic treatment, the protective effect of the commensal bacteria is decreased and increases the chance of invasion by bacterial pathogens. The most common disturbance of microflora results from the introduction of antimicrobial agents, antibiotics and medication, to the GI tract. Antibiotics reduce the types of bacteria in the GI tract, which allows for the growth of small populations of resistant bacteria (Wilson, 1997). When no longer kept in check by the predominant bacteria, these antibiotic resistant organisms can multiply and cause infection as they are typically more pathogenic than the bacteria which they are replacing (Wilson, 1997). Diarrhea is the most common symptom of GI infection. The ingestion of probiotic supplements has been demonstrated to lessen the duration of diarrhea (Isolauri and others, 1991; Kaila and others, 1992). 2.1.1 Lactic acid bacteria and bifidobacteria Lactic acid bacteria are Gram-positive bacteria that produce lactic acid as the major product of lactose fermentation. The following genera are generally considered typical LAB: Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. They are non-spore formers, highly acid tolerant, and grow best in a microaerophilic environment. LAB can be obligately homoferrnentative (producing only lactic acid as an end product) or facultatively heterofermentative (producing C02, acetic and lactic acids). LAB can change their metabolism depending on their growth conditions, but cannot compete with other bacteria in nutrient-poor conditions because they cannot produce all amino acids and vitamins necessary for their growth (Lucke, 1996). Lactic acid bacteria are among the microorganisms that make up the indigenous gut microflora. Due to the acidic conditions in the stomach, few bacteria inhabit the beginning of the GI tract. Lactobacilli can be found in the stomach at <103/g stomach contents (Salminen and others, 1998b) as some strains are more acid tolerant. Numbers of lactobacilli increase along the GI tract and typically reach between 104-109/g gut contents in the colon (Salminen and others, 1998b). Other genera of LAB found in the human large intestine are Enterococcus, and Streptococcus (Borriello, 1986). Bifidobacteria, formerly grouped in the genus Lactobacillus, make up an important population of the gut microflora. The number of bifidobacteria increases along the GI tract and reaches 108-1011/g gut content in the colon and can account for up to 10% of total flora and 25% of anaerobic strains (Mitsuoka, 1990; Salminen and others, 1998b). New molecular genetic techniques and chemotaxonomy developed in the 1960’s allowed scientists to recognize bifidobacteria as a unique group of bacteria. It was determined that bifidobacteria were genetically different from Lactobacillus, Corynebacterium and Propionibacterium because they had >5 0% G+C in the DNA, whereas LAB had <50% G+C (Holzapfel and Wood, 1998). Bifidobacteria are Gram-positive, strictly anaerobic, non-motile, non-spore forming bacteria. They are also unique in that they lack the enzymes aldolase and glucose-6-phosphate dehydrogenase needed for homo- and heterofementation. Instead, bifidobacteria degrade hexoses via the fi'uctose-6-phosphate pathway, of which, fructose- 6-phosphate phosphoketolase is the characteristic enzyme (Ballongue, 1993). Mitsuoka (1990) reported that bacterial composition of gut microflora changes with age (Figure 2.1). Bifidobacteria populations decrease or disappear with an increase in age, whereas populations of streptococci, enterobacteria, clostridia and lactobacilli increase. Clostridium perfringens, which is associated with gastroenteritis, significantly a— I!) hmEmm,Pmtomee-o g 0“ ‘~~~ ./.-— --------------- I—oo-o—o-o- 'OL .f.‘-------- ....... 3 / W " I E 8" : Eadnridriacofismbm / ' r 2 4 .’ _--,. 3 6 f' I l I l I W I. ‘ .q.. _'.—’.-I'_-o-Oo—oo-oo-l". /’ S if I l’ 8 ‘l‘ f i Wng d’/’ .J o ”—--——-—--d l k’ 2 babies mailings infants adutta aged Figure 2.1 Changes in human fecal flora with increase in age. (Reproduced from Mitsuoka, 1990). increases in the elderly. This alteration in microflora may make the elderly more susceptible to liver firnction disorders, pathogenic and toxic burdens, and cancer (Mallet and Roland, 1987). Bifidobacteria are able to maintain bacterial homeostasis in the gut with lactic and acetic acids produced during fermentation as well as production of other substances that are inhibitory to pathogens such as C. perfiingens and Escherichia coli (Gibson and Wang, 1994). 2.1.2 Probiotics Although earlier definitions describe probiotics as only having an affect in the gut, a definition that better fits recent studies was stated by Chandan (1999) as “strains of living microorganisms that on ingestion in certain doses exert health benefits beyond inherent basic nutrition.” Bacteria from the genera Lactobacillus and Bifidobacterium are the most commonly studied probiotic bacteria (Sanders, 1999). Probiotic cultures have been reported to have numerous health benefits, but only the alleviation of lactose intolerance symptoms and anti-diarrhea] effects have been substantiated through scientific studies (Marteau and others, 2001; Sanders, 1999). LAB are thought to produce lactase when in the presence of bile and then aid the digestion of lactose in the gut lumen (de Vrese and others, 2001). The administration of probiotics has been demonstrated to lessen the duration of acute rotavirus diarrhea. Children fed Lactobacillus casei sp strain GG had significantly shorter (1.1 d) bouts of diarrhea compared to 2.5 d for the control group (Kaila and others, 1992). It has been hypothesized that probiotics could also prevent or lessen diarrhea by colonization 10 resistance, adhering to intestinal mucosa and by blocking adherence by pathogenic bacteria or by influencing gut flora populations (Sanders and Huis in’t Veld, 1999). Additional health benefits attributed to probiotics include anti-cancer effects, reduced serum cholesterol, antihypertensive effects, stomach health (prevention of infection by Helicobacter pylori) and enhanced immune response (Fuller, 1991, Gilliland, 1990; Sanders, 1999). Probiotics have been implicated in reduced cancer risk because they may affect intestinal epithelial cell kinetics and decrease cancer cell proliferation in the colon (Sanders, 1999). In a study by A30 and Akazan (1992), L. casei increased the time between incidences of bladder cancer in humans. Two possible mechanisms by which probiotics could reduce serum cholesterol have been proposed. Probiotics may assimilate the cholesterol molecule or enzymatically deconjugate bile acids (Sanders, 1999). If probiotics do deconjuate bile acids, however, some could by converted to secondary bile acids which are cancer promoters. Antihypertensive effects have been attributed to tripeptides created from fermentation of milk by probiotics. These tripeptides acted as angiotensin-I—converting enzyme inhibitors and reduced blood pressure (Sanders, 1999). Results from animal and human trials indicate that probiotics and their end products such as lactic acid can prevent colonization by Helicobacter pylori. Colonization of the stomach by H. pylori has been reported to result in peptic ulcers, chronic gastritis and increased risk of gastric cancer (Marshall, 1994). With respect to enhanced immune response, probiotics could reduce cancer risk as well as have anti-infective activity. Currently, the mechanism of how probiotics exert these effects is unclear. ll 2.2 GI immune system The GI tract is made up of the stomach, small intestine and large intestine. Due to the exposure of these organs to foreign matter via ingested material, humans evolved with nonspecific and specific immune mechanisms for protection. The GI immune system plays an important role in the health of the individual. Nonspecific immunological defenses are intrinsic and include gastric acidity, small intestinal peristalsis, the indirect removal of bacteria by mucus and lysozymes and the gut microflora. To protect against antigens that survive these conditions, the host can launch a specific immune response that involves identification by lymphocytes, followed by proliferation and activation of additional immune cells (Pestka, 1993). Cells participating in the specific immune response include lymphoid follicles (Peyer’s patches), isolated follicles, mesenteric lymph nodes, intraepithelial lymphocytes (IEL) and the lamina propria (Shanahan, 1994) (Figure 2.2). Collectively, these cells are called the GALT. The intestinal epithelial cells give the first warning to underlying mucosa cells of bacterial invasion (Eckmann and others, 1993). More specifically, antigens from the gut lumen enter blood circulation via intestinal epithelial cells and Peyer’s patches (PP), which are groups of lymphoid follicles (Pestka, 1993). After antigen uptake and presentation, an immune response is mounted that leads to the production of 1g and cell- mediated immune responses. Epithelial cells in vitro secrete cytokines such as interleukin (IL)-6 (Hedges and others, 1992) and IL-8 (Eckmann and others, 1993), which are believed to influence the development of an immune response from leukocytes in the intestinal mucosa. Stimulation of IL-6 and IL-8 has been the focus of this research. 12 Intestinal - ~ VILLUS [PEYER'S J lntraepithelial PATCH Lymphocyte 3 T PROPRIA T GERMINAL CENTER Figure 2.2 Gut-associated lymphoid tissue (GALT). Peyer's patches and lamina propria are important elements of the intrinsic GALT. Location of immune cells are shown by the following abbreviations: B = B lymphocytes, T = T lymphocytes, Md) = macrophage, MC = mast cells. (Reproduced from Pestka, 1993). 13 2.2.1 Cytokines Cytokines are small non-antigen-specific protein molecules that cells use to influence each other. They work in a network where each cytokine may have multiple or overlapping functions (Playfair, 1996; Shanahan, 1994). Cytokines may work synergistically and stimulate the production of other cytokines. Depending on the effector cell type, they may have a harmful or a beneficial role in disease (Playfair, 1996) Two cytokines will be examined in this project: IL-6 and IL-8. IL—6 is a B cell differentiation factor that is needed for antibody secretion. It also plays a role in acute phase response and enhances inflammatory response (Akira and others, 1993). lL-6 is produced by a number of cell types including: T cells, B cells, smooth muscle cells, endothelial cells and monocytes/macrophages (Akira and others, 1993). In the case of malaria, increased IL-6 levels over a period of time have been associated with organ damage. Although it is associated with the pathology of diseases such as rheumatoid arthritis, multiple myeloma and acquired immunodeficiency syndrome (AIDS), it also has antitumor activities. IL-6, therefore, can act in an inhibitory or stimulatory manner depending on cell type. IL-8 is classified as a chemokine whose function is to attract T cells, monocytes and neutrophils to inflammatory sites (Playfair, 1996). Chemokines are a specific subset of cytokines characterized by a highly conserved sequence of four cysteines that influence their tertiary structures (Van Damme, 1994). IL-8 can protect blood vessel cells from neutrophil-mediated damage by inhibiting neutrophil adhesion to cytokine- activated endothelial cells (Gimbrone and others, 1989; Van Damme, 1994). High 14 amounts of IL-8, however, can stimulate adhesion of neutrophils to unactivated endothelial cells (Gimbrone and others, 1989). Increased IL-8 production also has been seen with infectious diseases of the central nervous system, gastric infection, ulcerative colitis and hemolytic uremic syndrome. 2.2.1 Effect of probiotics on immune nasponses in the GI gag Probiotics may increase non-specific immunity against tumors and infection as mentioned earlier. They may achieve specific immune responses by activating macrophages, increasing levels of cytokines and IgA, and by increasing the activity of natural killer (NK) cells (Sanders, 1999). Probiotics can exert these positive effects, but do not cause a harmful inflammatory response like some enteric bacteria perhaps because of the lack of lipopolysaccharide (LPS) in the cell wall. Although the exact mechanism by which probiotics exert their immune enhancing effects is not known, the ability to adhere to and colonize the intestine are thought to be important. Adherence allows the probiotics to be in close proximity to the GALT to have an effect and avoid ‘washing-out’ (Vaughan and others, 1999). Direct contact may be necessary for some immune effects such as enhanced leucocyte phagocyte activity against enterobacteria (Schiffrin and others, 1997). Colonization could ensure that the probiotics remained in the GI tract and continued to interact with the GALT. It is hypothesized that colonized probiotics exert protective effects by blocking attachment sites of pathogenic microorganisms and/or by steric hindrance (Tancrede, 1992). Probiotics also produce substances inhibitory towards other organisms. This capacity of 15 probiotics to produce bacteriocins and other antimicrobial peptides as well as the ability to alter pH, is defined as colonization resistance (Rolfe, 1996). Although strains of lactobacilli and bifidobacteria have been shown to adhere to human intestinal cells in vitro (Bernet and others, 1994; Chauviere and others, 1992; Crociani and others, 1995), this has not been confirmed in vivo. In the body, probiotics are challenged with stomach acid, bile and peristaltic movement of the intestine before they can have the opportunity to colonize the gut. Colonization of the gut by probiotics may, therefore, be temporary. In clinical studies by Lidbeck and others (1987), after administration of Lactobacillus supplements ceased, the levels of Lactobacillus in feces returned to pre-experimental levels. Continual intake of the probiotic may be necessary to achieve and maintain maximum numbers of bacteria. Other studies have shown, however, that probiotic bacteria may not need to be viable to exert an immunostimulatory response (Marin and others, 1997; Perdigon and others, 1986; Solis Pereyra and Lemonnier, 1993). It is possible that outer-membrane proteins of non-viable cells and/or their cell components may be all that is necessary to interact with receptors on GALT and provide an immune response. This would be similar to the immunostimulating effect of LPS, which is found on the outer membrane of Gram-negative bacteria. LPS can cause monocytes to secrete cytokines and also can potently activate B cells (Pestka, 1993). By contrast, some studies have compared live bacteria to nonviable cells and found that nonviable cells either did not produce an immune response or stimulated to a much lesser degree than the live cells (Haller and others, 1999; Miettinen and others, 1996). Due to the different models used for these 16 studies, firrther research is necessary to confirm whether viability is essential for probiotic function. 2.3 Immunostimulating effecg of lactic acid bacteria, bifidobacteria and milk W 2.3.1 In vitro studies One difficulty with intestinal studies is obtaining a reasonable model for the human GI tract. Due to the location of the intestinal tract in the body, human in vivo studies are not possible for most experiments. Therefore, most studies concerning probiotics and the immune system have used mouse models and various cell lines. Table 2.1 summarizes the recent in vitro studies on the immunostimulating effects of LAB, bifidobacteria and their cellular components. Miettinen and others (1996) used both live and glutaraldehyde-fixed LAB to stimulate human peripheral blood mononuclear cells (PBMC). Glutaraldehyde is a cross- linking agent that denatures proteins. They reported that live bacteria were better able to stimulate PBMC to secrete the cytokines IL-6 and tumor necrosis factor (TNF)-0t than glutaraldehyde-fixed bacteria. This effect, however, was strain specific. Although it is possible that the denaturation of proteins by glutaraldehyde altered their immune stimulating properties, Miettinen and others (1996) suggested that LAB should be viable to exert the optimal immunostimulating effect. Several other experiments, however, demonstrate the immunostimulating effects of heat-killed LAB and bifidobacteria as well as their cellular components. Tejada- Simon and others (1999a) demonstrated that whole cells of bifidobacteria and LAB, their 17 no... .8050 v.8 5...: mg. .82.... as. std—2 a... .85.. a... a... mag— .m.05o e5. Scam-0.8.5... a... .85.. a... as. 03. .8050 use .03.: 8... .22.... as. 3...... _ 8m .8056 E... 20>» 8.. :9. 8... .850 a... assuage n-.. e... 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Marin and others (1997) demonstrated that heat-killed bifidobacteria could enhance cytokine production by RAW 264.7 murine macrophage cells and EL-4.lL-2 thymoma cells (helper T-cell model). Incubation of fourteen different strains of bifidobacteria with RAW 264.7 cells significantly stimulated TNF-a and IL-6 production in a dose dependent manner. TNF-a and IL-6 production increased 21- to 872-fold and 9.3- to 204-fold, respectively, depending on strain. The addition of LPS tended to decrease the effect of bifidobacteria stimulation. Eight of the 14 strains of bifidobacteria significantly increased IL-2 production by EL-2.lL-4 cells at a concentration of 106 cells/ml. The effect of bifidobacten'a on IL-5 production by EL-2.IL-4 cells was more inconsistent. Bifidobacten'a were stimulatory or inhibitory depending on strain and concentration. Bifidobacteria Bf-6 and B. adolescentis Ml 01-4 were among the most stimulatory strains for all cytokines tested and therefore, were chosen for this study. Bf-6 is used in commercial dairy products. Park and others (1999) saw slightly varying results in RAW 264.7 cells stimulated with human and commercial isolates of bifidobacten'a. Bifidobacteria with the addition of LPS increased IL-6 production synergistically. The same combination, however, reduced TNF-a production. While all strains of bifidobacteria stimulated IL-6 and TNF- or production without LPS, strain dependent differences were observed. Compared to L. bulgaricus, B. adolescentis M1 01 -4 and Bifidobacterium Bf-6, S. thermophilus was even more effective at cytokine stimulation of RAW 264.7 and 20 EL4.1L~2 cells (Marin and others, 1998). Generally, S. thermophilus Stl33 had the most stimulatory effect on RAW 264.7 cells. Solis-Pereyra and Lemonnier (1993) also found S. thermophilus to be among the most stimulatory to lL-l B, TNF-a and interferon (IFN)- 7 production by human PBMC at a dose of 2 x 107 bacteria/2 x 106 PBMC. Viability may not be necessary for probiotics to stimulate immune function. For example, Marin and others (1997, 1998) and Park and others (1999) used heat-killed LAB to stimulate cytokine production in mouse cell lines. However, Haller and others (1999) used Lactobacillus sakei, Lactobacillus johnsonii strain La] and Lactobacillus paracasei strain Shirota at various stages of the cell cycle and observed a difference in cytokine production by human PBMC when stimulated with live and heat-killed bacteria. Live bacteria in the logarithmic growth phase were able to stimulate TNF-a production at a lower concentration than heat-killed bacteria from the same phase. However, heat- killed bacteria from the stationary growth phase were more effective at TNF-a stimulation than live bacteria also in stationary phase. Haller and others (1999) speculated that differences in bacterial growth phase were observed because the cell wall composition changes during growth and heat-inactivation may then affect these SU'UCIUTCS. 2.3.2 Animal studies Feeding studies in animal models also have demonstrated an enhanced immune response to oral administration of LAB. Recent studies with animal models are summarized in Table 2.2. Perdigon and others (1986) orally administered a mixture of Lactobacillus casei and Lactobacillus acidophilus (a total of 2.4 x 109 cfiJ/d) in non- 21 no... .32.... us. .32 33 «850 ES 5:8.»an 2.2 £050 new 295:0? 032 £05.. 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Digestive enzymes such as pepsin, trypsin and chymosin have ofien been used to synthesize bioactive milk peptides. Several studies have demonstrated that fermentation of milk by LAB can also release milk peptides with immunomodulating activities (Laflineur and others, 1996; McDonald and others, 1994). Table 2.4 describes recent studies of immunostimulation by milk components. Since bioactive milk peptides are formed in viva after the digestion of milk, Gill and others (2000a) suggested that they may help protect the neonatal bovine whose GI immune system has not yet fully developed. Several studies have examined the effects of bioactive milk peptides in vitro and in viva, though most studies have used in vitro methods. More research on the immunomodulating effects of milk peptides remains to be done as well as clinical trials to determine their effects on human health. Milk proteins are generally separated into two categories: caseins and soluble milk proteins. Table 2.5 lists bioactive milk peptides obtained from both types of milk proteins as well as their immune effects. 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Ease Esgm nausea—=8 use 3 song—Eamogeg me 868m 2.83— v." 03¢... 30 Table 2.5 Immune effects of bioactive milk peptides Milk protein precursor Bioactive peptide Immune effect BSA Serophin Opioid agonist (1,1 -CN Casecidin Antimicrobial activity Isracidin Antimicrobial activity c.1-Casokinin-5 ACE inhibitor Caseinophosphopeptide Calcium binding and Transport B-CN B-Casokinin-7 ACE inhibitor Antihypertensive peptide Antihypertensive peptide Caseinophosphopeptide hummostimulatory K-CN Casoplatelin Antithrombotic Casoxin C Opioid antagonist a-Lactalbmnin a-Lactorphin Opioid agonist ACE inhibitor B-Lactoglobulin B-Lactorphin Opioid agonist ACE inhibitor Lactoferrin Lactoferricin B Immunostimulatory Antimicrobial activity Lactoferroxin A Opioid antagonist lactonansferrin Lactoferroxins A, B, C Opioid agonist Adapted from Clare and Swaisgood (2000) and Schlimme and Meisel (1995) 31 (CN). Bioactive components of CN have demonstrated antihypertensive, antithrombotic, opioid, and immunostimulating effects. The immunostimulating effects include increased phagocytosis of human macrophages and protection against Klebsiella pneumoniae (Clare and Swaisgood, 2000). MacDonald and others (1994) used media containing CN digested by commercial yogurt cultures to determine if any of the end products of CN fermentation had an effect on colon cell kinetics. Their model included two intestinal cell lines: IEC-6 cells (fi'om normal rat intestine) and Caco-2 cells. Rates of [3H]thymidine incorporation and cell kinetics by flow cytometry were used to determine the effects of the bacteria-conditioned media on both cell lines. In general, [EC-6 cells had decreased rates of cell division, while Caco-2 cells demonstrated increased rates. Differences in cell divison were also observed between different starter cultures. Because a link has been observed between fermented milks and reduced risk of developing some cancers, a reduced rate of cell division could indicate a decrease in tumor development. The results from the two cell lines may differ because IEC-6 cells are normal, whereas Caco-2 cells are adenocarcinoma cells. Lafiineur and others (1996) also found that the effect of LAB fermented casein was strain dependent. Ten LAB were grown in ultrafiltration permeate of bovine milk supplemented with B-CN as the protein source. Supernatant from this digest was examined for its immunologic effects on human PBMC. Only supernatant from Lactobacillus helveticus 5089 caused lymphocyte proliferation from all blood donors. When PBMC were stimulated with the mitogen ConA, L. helveticus supernatant inhibited cytokine IL-2 production compared to the control sample which was not 32 fermented. Conversely, IFN-y production was increased by ConA-stimulated PBMC. Lafiineur and others (1996) suggest that some peptide formed from B-CN digestion by LAB can interact and stimulate proliferation of lymphocytes by increasing cytokine secretion. Some soluble milk proteins and bioactive peptides obtained by their digestion have demonstrated immunomodulating activity. Soluble milk proteins include a- lactalbumin (or-la), B-lactobglobulin (B-lg), bovine serum albumin (BSA), 1g, lactoferrin and lactoperoxidase (Horton, 1995). Alpha-la and [Hg are currently used to supplement speciality foods such as infant formulas and sports and dietetic beverages (Horton, 1995). The bioactive peptides, a-lactorphin and B-lactorphin, obtained from digestion of a-la and B-lg, respectively, are agonist peptides with morphine-like activity (Clare and Swaisgood, 2000; Schlimme and Meisel, 1995). Wong and others (1998) examined the effects of purified bovine milk proteins on murine spleen cells. They found that B-lg alone significantly increased cell proliferation and production of IgM compared to CN, and mixtures of a-la, B-lg, BSA, and bovine gamma globulin (BGG). Alpha-la, BSA and B66 alone did not stimulate IgM production, but the data were not shown. When B-lg was treated with alkaline or digested with trypsin, stimulation of IgM was greatly diminished. Wong and others (1998) found their results to conflict with other studies which determined that BSA was the most immunostimulatory bovine whey protein (Bounous and others, 1989; Bounous and Kongshavn, 1985). They suggested that their experimental model may account for the difference in results, but they also could not rule out the possibility of a copurifying substance in the [Hg 33 2.4 Caco-2 cells More recently, Caco-2 cells have been used as an in vitro model for human intestinal epithelial cells because of their physical and functional similarities. Caco-2 cells are enterocyte-like colonic adenocarcinoma cells, which are hypertetraploid. They are able to spontaneously differentiate in culture and form tight junctions, and thus resemble normal intestinal epithelial cells. Caco-2 cells also exhibit structures resembling brush border microvilli. As to why Caco-2 cells, isolated from the colon, are able to differentiate into enterocytes is still not well understood. Pinto and others (1983) speculated that Caco-2 cells have several chemical characteristics similar to fetal cells, which undergo differentiation. Caco-2 cells are thought to be good models for immune studies because they secrete cytokines. Jung and others (1995) compared cytokine production from freshly isolated normal colon epithelial cells and colon epithelial cells lines. Freshly isolated epithelial cell production of monocyte chemotactic protein-1 (MCP-l, a chemokine) and ILL-8 was upregulated by bacterial invasion or by IL-IB stimulation in the same order of magnitude as Caco-2 cells. Caco-2 cells produced mRN A for the cytokines TNF-or, IL-8, MCP-l and granulocyte-macrophage colony-stimulating factor (GM-CSF) as a result of infection with invasive bacteria such as Salmonella dublin, Shigella dysenteriae and L. monocytogenes (Jung and others, 1995). In the case of lL-6, freshly isolated epithelial cells produced increased amounts of this cytokine from bacterial stimulation, whereas Caco-2 cells did not. From this study, it appeared that the Caco-2 cell line did not have the same response to stimuli as fresh epithelial cells. This comparison is important because cell lines are typically homogeneous and easier to grow in culture than fresh 34 cells. Several contrasting studies have shown that Caco-2 cells can produce IL-6 and IL- 8 after exposure to bacterial pathogens (Michalsky and others, 1997; Eckmann and others, 1993; Hedges and others, 1992). Table 2.6 gives a summary of these and other studies of cytokine production by Caco-2 cells. Vitkus and others (1998) found that Caco-2 cells were capable of producing IL-6 when stimulated with cytokines IL-lB and TNF-a. Caco-2 cells grown in 5% C02 produced significantly lower (p<0.05) amounts of IL-6 when stimulated with either IL-1 [3 or TNF-a, as compared to those grown in 10% C02. Caco-2 production of IL-6 by independent stimulation with IL-lB and TNF-a was dose dependent; but co-stimulation resulted in a synergistic effect. Vitkus and others (1998) suggested that unstimulated Caco-2 cells were previously thought to be incapable of producing IL-6 because they were incubated in 5% CO; Caco-2 cells can therefore produce cytokines as a result of stimulation by other cytokines as well as bacterial pathogens. Based on the ability of Caco-2 cells to secrete IL—6, IL-8, and TNF-a, Vitkus and others (1998) concluded that Caco-2 cells make an excellent model for normal intestinal epithelial cell cytokine stimulation. The use of Caco-2 cells as a model to investigate the immunostimulating effects of probiotics has been limited. Studies have primarily focused on adhesion of LAB to Caco-2 cells (Tuomola and Salminen, 1998; Bernet and others, 1994; E10 and others, 1991). 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Using a transwell cell culture system, Caco-2 cells were incubated in separate but adjoining compartments which contained human PBMC (Figure 2.3). Non-pathogenic bacteria were not able to induce chemokine 1L-8 or MCP-l mRNA from Caco-2 cells alone. When Caco-2 cells and PBMC were cultured together, however, expression of mRN A for IL-8 and MCP-l was observed. E. coli and L. sakei also had a stimulatory effect on PBMC-sensitized Caco-2 cells to produce the cytokines TNF-a and H.-1B which followed the same trend as mRN A expression. L. johnsonii did not stimulate production of TNF-a or IL-lB and also did not induce as much mRN A of IL-8 or MCP-l as L. sakei. L. johnsonii did, however, stimulate transforming growth factor-6 in Caco-2 cells. Haller and others (2000) concluded that immunocompetent cells were necessary for Caco-2 cells to recognize non- pathogenic bacteria. Their communication was thought to be through soluble factors since Caco-2 cells and PBMC were not in direct contact in the transwell culture plates. 37 ( Apical compartment < Caco-2 cells t l Semi-permeable _. l l I T 1 L I I I Culturemedium membrane / f Basolateral compartment fl PBMC k Bottom of the plate Well Figure 2.3 Schematic diagram of the transwell co-culture system used by Haller and others (2000). Caco-2 cells were grown on a cell culture insert and then placed in the apical compartment. They were separated from the peripheral blood mononuclear cells (PBMC) (2 x 106 cells/ml) in the basolateral compartment by a semi-permeable membrane. Both wells were filled with culture media. 38 2.5 Rationale for this research Many questions concerning the immunomodulating effects of probiotics on the human immune system remain to be answered. Research needs to address if and how probiotics exert their effects. The limited number of studies investigating the interaction of probiotics and the immune system is due in part to the difficulty in finding an appropriate model system. To look at this interaction, the Caco-2 cell line was chosen because it is a well-established human cell line with similarity to epithelial cells. The working hypothesis for this research was that LAB and bifidobacteria, which are used in the production of fermented dairy foods, can enhance immune function by stimulating cytokine secretion by intestinal epithelial cells. The probiotic bacterial strains were chosen based on their ability to stimulate cytokine secretion in murine macrophage and T-cells. Because nutritional composition of milk changes during fermentation, probiotic bacteria were allowed to ferment reconstituted non-fat dry milk. Prior to use in the cell culture system, bacterial cultures were also inactivated by heat or by irradiation to compare if the immune effects seen by heat-treated cells were due to changes in cellular proteins. Further studies were conducted to determine the effects of milk components on cytokine secretion. 39 CHAPTER 3 MATERIALS AND METHODS 40 CHAPTER 3 MATERIALS AND METHODS 3.1 Culture preparation Seven probiotic organisms were selected based on their ability to stimulate cytokine production in previous experiments with murine macrophage and thymoma cell lines (Table 3.1) (Marin and others, 1997; Marin and others, 1998; Tejada-Simon and others, 1999). Lactobacillus acidophilus LA2 (LA), Lactobacillus delbril‘ckz'i subsp. bulgaricus (hereafier referred to as Lactobacillus bulgaricus) NCK 231 (LB), Lactobacillus casei ATCC 39539 (LC), and Lactobacillus reuteri ATCC 23272 (LR) were grown in De Man, Rogosa, Sharpe (MRS) broth (Difco Laboratories, Detroit, MI). Streptococcus thermophilus St 133 (STl33) was grown in M17 broth (Difco). Bifidobacterr‘um Bf-6 (BF6) and Bifidobacterium adolescentis M101-4 (M1014) were grown 'in MRS broth with 5% lactose under anaerobic conditions (GasPak®, BBL Microbiology Systems, Cockeysville, MD). In preliminary experiments, standard curves of optical density (OD) vs CFU/ml were generated for each bacterium based on spectrophotometric and plate count methods. ODs of cultures in their respective broths were measured on a Spectronic 1001 Plus (Milton Roy, Rochester, NY) at 650 nm using uninoculated broth as a blank. Culture samples were then diluted and plated to correlate cell numbers with OD. Bacteria in broth were diluted using 0.1% bacto-peptone dilution buffer (Difco) to obtain ten-fold dilutions of 10" to 10'8 CFU/ml. One ml samples were plated using the pour plate method. Lactobacilli, streptococci and bifidobacteria were enumerated using MRS, M17 broth, and MRSL containing 1.5% agar, respectively. Lactobacilli and streptococci 41 5 £83? 3.58355 5.8 m: .m assuage ”saucepan a: 6:303. 582.8 2330 25. 53:82 ~28 8.2 $32 9.5293293 a: 6:302 580:8 23.5 25. 585.5, 3% 8:, $8 33223 02 £325— éegeo 23m «535 582 EN 202 caress .33833 5 38%? 533235 :23 «-3 assesses sausages 59: .938. don—€53 265m 5&2. 75:2 mazeouéohe Sataoeaefixfi 5 28%? 5382.65 macaw Em §E§€§m 083m 585 «5.8m 3:: a... a. :3... 853.3 $.82:— 28 38a. E... 3.29235.— ..e 8958 fin 033—. 42 plates were incubated at 37°C for 48 h and then counted using a Darkfreld Quebec Colony Counter (American Optical Company, Bufi'alo, NY). Bifidobacteria were incubated anaerobically using the GasPak® system under the same conditions before being counted. Figure 3.1 provides a schematic diagram of sample preparation. One ml of each of the stock cultures (stored at —80°C) was thawed before being added to 25 ml of their respective broths as mentioned above. The cultures were then incubated for 12 h at 37°C at which point they had reached late log phase of growth. After incubation, 5 ml of inoculum was transfered to 25 ml of fresh broth and incubated for 12 h at 37°C. This transfer was repeated 2 more times before cultures were prepared for use in cell culture. After the third transfer, all probiotic cultures were harvested at late log phage (12 h). ODs were taken of the cultures to determine cell concentrations using the standard curves. The cultures were then centrifuged at 3000 x g for 15 min. The supernatant (broth) was discarded and cultures were washed with phosphate buffered saline (PBS) by centrifugation (3000 x g, 15 min) and decanted. The cultures were then resuspended in 10% reconstituted non-fat dry milk (NFDM) (Difco) to obtain final concentrations of 10°, 107, and 10° CFU/ml according to calculations using the standard curves. Plate counts of bacterial samples were performed before inactivation to confirm cell numbers using the pour plate method as described previously. Heat or irradiation was used to inactivate the prepared cultures. For heat- inactivated samples, bacteria were either heated (95°C, 30 min) immediately after preparation or after fermentation (3 7°C, 4 h), where cell numbers increased one log. The cultures at 108 CFU/ml were not fermented because the acid produced lowered the pH 43 . Bifidobacterr'um Bf-6 . Bifidobacterium adolescentis M101-4 . Streptococcus thermophilus St 133 . Lactobacillus bulgaricus NCK 231 . Lactobacillus acidophilus LA2 . Lactobacillus casei ATCC 39539 . Lactobacillus reuteri ATCC 23272 \JONMthNt—n 1 ml of each culture from frozen stock Suspend in 25 ml of broth: lactobacilli in MRS; streptococci in M17; bifidobacteria in MRSL. Incubate (37°C, 12 h) X 3 5 ml of inoculum 25 ml of respective bro . Incubate (37°C, 12 h) Read 0 at 650 nm Centrifuge (3000 x g, 15 min) Wash with PBS Centrifuge (3000 x g, 15 min) Resuspend in 10% NFDM I f l 106 10’ 108 cells/ml cells/ml cells/ml NF F NF F NF HK HK HK HK HK 01‘ or 01’ 01' or I 1 1 1 I Figure 3.1 Schematic diagram of probiotic culture preparation in non-fat dry milk (NFDM). MRS = DeMan, Rogosa, Sharpe; MRSL = MRS + 5% lactose; OD = optical density; NF = non-fermented; F = fermented (37°C, 4 h); HK = heat-killed (95°C, 30 min); I = irradiated (l Mrad). 44 and caused the NFDM to coagulate. This made administering a uniform to the cell culture difficult. Samples were then frozen at -80°C until further use. For irradiated samples, bacteria were either frozen (-80°C) immediately after preparation, or after fermentation (3 7°C, 4 h). Frozen samples were exposed to 1 Mrad of cobalt-60 irradiation at the University of Michigan Memorial Phoenix Project (Ann Arbor, MI). Irradiated samples were stored frozen at -80°C until further use. Appendix I contains a certificate of compliance for irradiation of LAB and bifidobacteria. Plate counts of probiotic cultures were performed before their addition to cell culture to verify cell numbers using the pour plate method as described above. 3.2 Caco-2 cell culture Caco-2 cells (ATCC HTB-37) were obtained from American Type Culture Collection (Rockville, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA), 0.01% (v/v) antibiotic-antimycotic solution (10.0 units/m1 penicillin G sodium, 10.0 ug/ml streptomycin sulfate, and 25.0 ug/ml amphotericin B in 0.85% saline) (Gibco), 0.01% (v/v) Fungizone reagent (Gibco), and 0.004% (w/v) sodium bicarbonate. Caco-2 cells were first grown in 25 cm2 tissue culture flasks at 37°C and 6% C02. Cells were loosened from the flask using Trypsin-EDTA (Sigma, St. Louis, MO) and harvested by centrifugation at 1200 x g for 7 min. Cells were transferred to 48-well tissue culture plates (Costar, Cambridge, MA) at 5 x 105 cells/well. Cell numbers were determined using a Bright-line hemocytometer (American 45 Optical Co., Buffalo, NY). Monolayers of Caco-2 cells were incubated for 72 h until confluent before use in experiments. 3.3 Stimulation of cytokine production of Caco-2 cells by probiotic bacteria Heat-inactivated or irradiated probiotic samples in NFDM as described in section 3.1 were added to a monolayer of Caco-2 cells at final concentrations of 10°, 10', and 108 cells/ml in a well. Uninoculated NFDM and the Caco-2 supernatant alone were used as negative controls. The cytokine IL-IB (1 ng/ml) was used as a positive control for IL-6 and IL-8 induction. Supernatant was collected at 24 and 48 h and frozen at -80°C until analyzed for the cytokines IL-6 and IL-8 by enzyme-linked immunosorbant assays (ELISA). 3.4 lL-6 and IL—8 quantitation Procedures included in the OptEIATM Set (BD PharMingen, San Diego, CA) were followed for the ELISA. Briefly, 100 pl of anti-human IL-6 or IL-8 monoclonal antibodies diluted in 0.1 M sodium carbonate buffer (pH 9.5) was added to each well of microtiter strips (Immunolon II Removawell; Dynatech Technologies, Chantilly, VA) set in a Removawell holder (Dynatech Technologies). The plates were incubated overnight at 4°C. Wells were then washed 3x with 0.01 M PBS with 0.05% Tween-20 (v/v) (PBST) using the Ultrawash Plus ELISA washer (Dynatech Technologies) to remove unbound capture antibody. The plates were then incubated for 1 h with 200 u] of PBS buffer supplemented with 10% FBS (v/v) (pH 7.0) to reduce nonspecific binding. Next, the wells were washed 3 x with PBST before 100 pl of standards of recombinant human IL-6 or IL-8 diluted with DMEM with 10% NFDM or sample were added to the wells. Plates were covered with aluminum foil and incubated at room temperature (~24°C) for 2 h. The wells were next washed 5 x with PBST to remove non-adhering antigens. One hundred ul of biotinylated anti-human IL-6 or IL-8 streptavidin-horseradish peroxidase conjugate (BD PharMingen) was added to each well and incubated at room temperature (~24°C) for 1 h. The wells were then washed 7x with PBST before 100 ul of tetrarnethylbenzidine substrate reagent (BD Pharmingen) was added to the wells. The plates were incubated at room temperature (~24°C, 30 min) in the dark. To stop the enzyme reaction, 50 u] of 2 N H2804 stopping solution was added to each well. Absorbance was read at 450 nm using a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA). Cytokines were quantitated using a standard curve generated from the Sofimax curve-fitting program (Molecular Devices). Higher absorbence readings indicated greater amounts of cytokine present in the supernatant sample. Cytokine values were expressed as percent change from Caco-2 supernatant which was designated as 100%. Values were calculated as follows: 100- (100*((cytokine in Caco-2 supematant-cytokine in sample)/cytokine in supernatant)). 3.5 Stimulation of cytokine production of Caco-2 cells by milk components Lactose (Sigma-Aldrich, St. Louis, MO), ct-la (Sigma-Aldrich), B-lg (Sigma- Aldrich) and NFDM were suspended in DMEM to obtain a 4% final concentration in cell culture. All solutions were filter sterilized using a 0.45pm Millex®HA syringe driven filter unit (Millipore Corporation, Bedford, MA). Solutions containing the milk 47 components were added to a monolayer of Caco-2 cells. After 2 h of incubation, IL-IB (1 ng/ml) was added to half of the samples. All samples were incubated (37°C, 6% C02) for 24 h. Supernatants were collected and frozen at —80°C until analyzed for the cytokines IL-6 and IL-8 by ELISA as described in section 3.4. The IL-6 and IL-8 standards, however, were suspended in DMEM for these experiments. 3.6 Statisitical analysis Experiments were replicated three times in a randomized design. Percent change of IL-6 and IL-8 production relative to the Cece-2 cell supernatant (calculation described in section 3 .4) was transformed by square root to correct for non-normality and heterogenous variances among samples. The data was analyzed using ‘PROC MIXED’ in The SAS system version 8.2 (SAS Institute Inc, 2001, Cary, NC). The model accounted for interaction between replication, probiotic organism, treatment, inactivation, concentration and plate. The main effects were probiotic organism (listed in Table 3.1), treatment (fermented or non-fermented), inactivation (heat or irradiation) and concentration (10°, 107, 108 CFU/ml). Two-way interactions of these main effects were also examined (i.e. organism*treatment, orgarrism*inactivation, etc.) Replication and plate were considered random effects. Significance of the main effects was tested using Type 3 sums of squares. The Satterthwaite degrees of freedom method was used with the Tukey-Kramer adjustment was conducted for multiple comparisons. A p50.05 was used as the level of significance. 48 CHAPTER 4 RESULTS AND DISCUSSION 49 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Effect of lactic acid bacteria and bifidobacteria on IL-6 production by Caco-2 cells Heat-killed and irradiated lactic acid bacteria and bifidobacteria were incubated with a monolayer of Caco-2 cells for 24 and 48 h. The supernatants were collected and then analyzed for the cytokines IL-6 and IL-8 using ELISA. Due to the differences in baseline cytokine production between replications, percent change in cytokine production was calculated where the amount of cytokine in the Caco-2 DMEM supernatant alone was 100 percent. When stimulated by IL-lB, the positive control, Caco-2 cells were capable of producing 2 to 11 times more 1L-6 than untreated controls. Uninoculated, reconstituted NFDM alone served as a negative control and did not significantly affect IL-6 and IL-8 production by Caco-2 cells during the 24 and 48 h incubations. The ‘PROC MIXED’ statistical procedure was used to determine the significance of each main effect interaction (i.e. culture by fermentation, culture by method of inactivation). The data were organized so that two sets of comparisons could be made. The first set accounted for all non-fermented samples at 10°, 10', and 108 CFU/ml, with the exception of ST133 which could not be grown to 108 CFU/ml. A separate analysis that did include ST133 indicated that it was not significantly different (p>0.05) from the other probiotic organisms at concentrations of 10° and 107 CFU/ml for any of the effects tested. The second set of data compared all fermented and non-fermented samples at 10° and107 CFU/ml. Standard errors of the means were not included in the figures because 50 they were generated based on the square root of the data. This was done to normalize the data. 4.1.1 Effect of culture When comparing the non-fermented samples only, the type of culture was not significant (p>0.05) to IL-6 production at 24 h (Table 4.1), but became significant (p<0.05) at 48 h (Table 4.2). The pooled concentrations of BF6 stimulated significantly more IL-6 (112.6%) than LB (82%), LC (90.7%), and M1014 (89.2%) (Figure 4.1) at 48 h. LA stimulated significantly greater IL-6 (99.2%) than LB (82%). Stimulation of IL-6 by NFDM was only significantly different (110.5%) from LB. Levels of IL-6 stimulated by LR and ST133 were not significantly different from any other the other cultures. Although it was not significant (p>0.05), NFDM and BF6 stimulated more IL-6 compared to the Caco-Z supernatant. With the exception of BF6, the addition of culture to NFDM suppressed IL-6 production. LB was the only culture which significantly suppressed (p<0.05) IL-6 production compared to the supernatant from naive Caco—2 culture. Comparing the fermented and non-fermented samples, the type of culture was not significant (p>0.05) to IL-6 production at 24 (Table 4.3) and 48 h (Table 4.4). 4.1.2 Effect of dose Dose was not significant to IL-6 production at either incubation time point. None of the concentrations were significantly different from one another. It cannot be concluded however, that there was no dose effect on IL-6 production by Caco-2 cells 51 Table 4.1 ANOVA table comparing non-fermented cultures at concentrations of 10°, 10’, and 10° CFU/ml on Ill-6 production by Caco-2 cells after 24 hr incubation Effect Degrees of F Value Pr > F Freedom Culture‘ 715 1.55 0.1486 Concentration2 71 1 1 .91 0. 1485 Inactivation3 715 60.9 <0.0001 Culture*Concentration 71 l 0.43 0.9589 Culture*Inactivation 71 0 4.99 <0.0001 Inactivation*Concentration 71 1 0.45 0.6387 l Probiotic cultures 2 106, 107,108 CFU/ml 3 Heat-killed/Irradiated 52 Table 4.2 ANOVA table comparing non-fermented cultures at concentrations of 10°, 107, and 10° CFU/ml on IL-6 production by Caco-2 cells after 48 hr incubation Effect Degrees of F Value Pr > F Freedom culture‘ 703 4.67 «1.0001 Concentrationz 710 0.53 0.5861 Inactivation3 716 148.1 F Freedom Culture1 1009 1.85 0.0748 Fermentation’ 1010 5.71 0.0171 Concentration’ 1003 1.01 0.3151 Inactivation“ 1007 48.5 <0.0001 Culture*Fermentation 1005 2.50 0.0152 Culture*Concentration 1003 0.93 0.4808 Culture*Inactivation 1010 2.53 0.0140 Fermentation'Concentration 1004 0.27 0.6030 Fermentation*Inactivation 1014 2.09 0. 1483 Inactivation*Concentration 1004 0.94 0.3 3 15 ; Probiotic cultures 3 F egmen7ted/Non-fermented 4 10 , 10 CFU/ml Heat-killed/Irradiated 55 Table 4.4 ANOVA table com aring fermented and non-fermented cultures at concentrations of 10° and 10 CFU/ml on IL-6 production by Caco-2 cells after 48 hr incubation Effect Degrees of F Value Pr > F Freedom Culture‘ 1011 1.04 0.4001 Fermentationz 1015 0.26 0.6118 Concentration3 1008 0.56 0.4537 Inactivation“ 1013 172.84 <0.0001 Culture*Fermentation 1010 1 .82 0.0799 Culture*Concentration 1008 0.98 0.4422 Culture*Inactivation 1013 1.96 0.0572 Fennentation*Concentration 1008 0. 1 9 0.661 5 Fermentation*lnactivation 1016 0.00 0.9506 Inactivation*Concentration 1008 0. 1 5 0.7026 ; Probiotic cultures Fermented/Non-fermented 3 10", 107 CFU/ml 4 Heat-killed/Irradiated 56 L. because no single concentration stimulated or suppressed significantly different (p>0.05) amounts of IL-6 compared to the Caco-Z supernatant based on the confidence intervals. 4.1.3 Effect of fermentation The effect of fermentation by probiotic bacteria on IL-6 production by Caco-2 cells was examined at doses of 10° and 107 CFU/ml. After 4 h incubation, cell numbers increased by one log. Ferrnentaticn of NFDM by probiotic bacteria had a significant effect (p<0.05) on IL-6 production by Caco-2 cells after 24 h incubation (Table 4.3), but this was no longer seen at 48 h (Table 4.4). Compared to the Caco-2 supernatant control, non-fermented samples suppressed IL-6 production to 96.5%. This was statistically higher than 90.9% lL—6 stimulated by fermented samples. Suppression of IL-6 production compared to the naive Caco-2 supernatant, however, was not significant (p>0.05). These results indicate that fermentation yielded end products which were slightly inhibitory to IL-6 production by Caco-2 cells. 4.1.4 Effect of inactivation The difference in IL-6 production between heat-killed and irradiated culture samples was significant (p<0.05) upon comparison of all non-fermented samples at 24 and 48 h (Tables 4.1, 4.2). More specifically, at 24 h, heat-killed samples stimulated IL-6 production to 106.9% control, whereas irradiated samples suppressed IL—6 production by 15.2%. None of the samples produced significantly (p<0.05) different levels of IL-6 compared to the Caco-2 supernatant. At 48 h, the same trend was seen where heat-killed 57 samples stimulated IL-6 production (114.6%), but in irradiated samples IL-6 production was suppressed (81%). The comparison of fermented and non-fermented culture samples also showed that method of inactivation resulted in significantly different (p<0.05) levels of IL-6 production. At 24 11, irradiated cultures suppressed lL-6 production to 86%, which was significantly lower (p<0.05) than the heat-killed cultures (101.7%). At 48 h, heat-killed cultures stimulated more 1L-6 (116.1%) than the irradiated cultures (82.4%). These results suggest that the mode of inactivation is significant to the amount of cytokine produced. It is possible that the exposure to heat denatured proteins on the bacterial surface or in the cytoplasm which stimulated IL-6 production. Future studies may want to use irradiated cells to ensure more accurate results. 4.1.5 Effect of the intermmmM—W The interaction between culture and dose was statistically significant (p<0.05) when comparing IL-6 production in the non-fermented cultures at 48 h (Table 4.2). Upon closer examination, however, the individual interactions were between different cultures, not within a culture. We were only interested in comparison between different doses of the same strain to determine if there was a dose effect on IL-6 production. 4.1.6 Effect of the interaLction between culture and inactivation The interaction between culture and inactivation was significant at both time points for the non-fermented samples (Tables 4.1, 4.2). Figure 4.2 illustrates the effect of culture and inactivation on IL-6 production by Caco-2 cells at 24 h. With the 58 140 - Heat-killed * 120 1 . , Irradiated * g is '3 100 3 "1 O ,— 7“: ;; '7' 77 a 80 1 :9} r E; if? ‘3 ” *2; - 5?: ’1 d «E 60 4 1‘3 51 if 0 . g 5:: iii irf ;.;1 is" U 40 1 3?: 33’: 191 :91 3: ~,:. ..\° {:5 _?j3 1: 20 - 1 5:; .fl 1 0 r ' NFDM LA LB LC LR ST133 M1014 BF6 Culture Figure 4.2 Effect of heat and irradiation inactivation of non-fermented lactic acid bacteria and bifidobacteria at concentrations of 10°, 107, and 10° CFU/ml on IL-6 production by Caco-2 cells (24 h incubation). Unstimulated IL-6 in Caco-2 supernatant was considered 100 percent. "' Indicates that heat-killed are significantly different than the irradiated counterpart (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgaricus NCK 231; LC = L. casei ATCC 39539; LR = L. reuteri ATCC 23272; ST133 = S. thermophilus Stl33; M1014 = B. adolescentis M101-4; BF6 = Bifidobacterium Bf-6 59 exception of NFDM, all heat-killed cultures caused Caco-2 cells to produce approximately the same amount of IL-6 compared to the Caco-2 supernatant. Irradiated cultures however, with the exception of NFDM, suppressed IL-6 production although it was not a significant amount (p>0.05) from the naive Caco-2 supernatant. The heat- killed samples of BF6 and LB stimulated significantly higher levels of IL—6 (p<0.05) than their irradiated counterparts. None of the cultures when incubated with the Caco-2 cells resulted in IL-6 production that was significantly different from the naive Caco-2 cells. Figure 4.3 demonstrates that at 48 h, the heat-killed and irradiated cultures showed the same trends on IL-6 production by non-fermented cultures as at 24 b (Figure 4.2). At this later time point, however, there was a significant difference (p<0.05) between the heat-killed and irradiated cultures of LA, LB, LC, LR and BF6. The interaction of LB, LC, LR, and M1014 and irradiation resulted in suppression of IL-6 production that was significantly lower than the IL-6 induction by the naive Caco-2 cells. Comparing the interaction between culture and inactivation for fermented and non-fermented cultures indicated that this interaction was only significant at 24 h. Figure 4.4 illustrates that irradiated cultures suppressed IL-6 production by Caco-2 cells to a much greater extent than heat-killed cultures. There was a significant difference (p<0.05) between the irradiated and heat-killed cultures of LC, M1014 and BF6. None of the cultures resulted in IL-6 production that was significantly different from the Caco-2 supernatant. The difference between heat- and irradiation-inactivation was significant for several of the cultures, however this was not consistent between time points or comparisons. Heat-inactivation resulted in substances that were not as inhibitory to IL-6 60 - Heat-killed 140 ~ Irradiated * t * 120 '1 * a .2 2:; g» ‘°° 1'1 w II: «9 8° :2 d 1- 11 .E 60 _ $31 1 a, a. 451 11? g f; EQ: 1f; .1: QE‘ 0 4° 1 I“ ,5: e\° .f if 2;: i: ,.: iii 1:; 2° 1 ii? f f ‘1 .f 5.; _;; ., .1 1 1. 0 I , . . _ .. I NFDM LA LB LC LR ST133 M1014 BF6 Culture Figure 4.3 Effect heat and irradiation inactivation on non-fermented lactic acid bacteria and bifidobacteria at concentrations of 10°, 107, and 10° CFU/ml on IL-6 production by Caco-2 cells (48 h incubation). Unstimulated IL-6 in Caco-2 cell supernatant was considered 100 percent. * Indicates that heat-killed were significantly difierent than irradiated counterpart (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgaricus NCK 231; LC = L. casei ATCC 35935; LR = L. reuteri ATCC 23272; ST133 = S. thennophilus Stl33; M1014 = B. adolescentis M101-4; BF6 = Btfidobacterium Bf-6 61 120 - - Heat-killed . "35727513'5'1 Irradiated 100 =3 "' .9 =- Q .5: “3 =3 60 4 .5 8* . g 40 4 U O\° {3 5:; 20 4 1 0 [.12 [:4 I" #71 (fir-.Vl—F‘ rfimil‘slt‘li'livr .. ...4-..... .3... .. ”4... .',.-... '.. NFDM LA LB LC LR Culture Figure 4.4 Efl‘ect of heat and irradiation inactivation of fermented and non- fermented lactic acid bacteria and bifidobacteria at concentrations of 106 and 107 CFU/ml on IL-6 production by Caco-2 cells (24 h incubation). Unstimulated IL-6 in Caco-2 supernatantwas considered 100 percent. * Indicates that heat-killed were significantly different from irradiated counterpart (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgaricus NCK 231; LC = L. casei ATCC 39539; LR = L. reuterr' ATCC 23272; ST133 = S. themophilus Stl33; M1014 = B. adolescentis M101—4; BF6 = Bifidobacterium Bf-6 62 ST133 M1014 BF6 production by Caco-2 cells than irradiated cultures. 4.1.7 Effect of the interlaction between culture_ and fermentation The interaction between culture and fermentation was significant (p<0.05) at 24, but not at 48 h. Upon closer examination, however, the specific interactions that were significant were not between the same culture and therefore not a valid comparison for this study. It can be concluded that fermentation did not make a significant difference in [ls-6 production for any of the cultures studied. 4.1.8 Discussion on the effect of lactic acid bacteria and bifidobacteria on IL-6 production by Cage-2 cells In general there were no consistent culture- or dose-dependent observations relative to IL-6 production by Caco-2 cells. None of the cultures significantly stimulated (p>0.05) 115-6 production at any dose which is contrary to the findings of Marin and others (1998), who examined the effect of LAB and bifidobacteria on cytokine production by mouse RAW 264.7 macrophage cells and mouse EL4.IL-2 thymoma cells. In the latter study, all bacteria were heat-killed and incubated with cell lines at concentrations of 10°, 107, and 10° bacteria/ml. Probiotic strain- and dose-dependent increases were observed with respect to IL-6 and TNF-a production by RAW 264.7 cells as well as in IL-2 and IL-5 production by EL4.IL-2 cells. Compared to other bacteria studied, S. thermophilus ST133 (also used in our experiments) had the greatest enhancing effects on cytokine production. In general, they observed that as the concentration of all bacteria increased, so did the amount of cytokine produced by the respective cell lines. 63 There are other reports of strain and concentration effects of probiotics and their components on cytokine production (Park and others, 1999; Tejada-Simon and Pestka, 1999; Miettinen and others, 1996). Our results also differed from these previous studies, possibly because the cell models differed. It is also possible that Caco-2 cells may require communication with underlying immune cells in order to respond to Gram- positive bacteria. Haller and others (2000), as mentioned in section 2.7, used Caco-2 cells in a co-culture system with human blood leukocytes where the two types of cells were separated by a membrane in transwell culture plates. Without the leukocytes, Caco- 2 cells could not be stimulated by L. sakei to produce cytokines TNF-a and IL-lB. Transfer experiments were performed using leukocyte-sensitized Caco-2 cells to determine if it was the Caco-2 cells or leukocytes responsible for cytokine production. After a 12 h initial incubation with leukocytes, Caco-2 cells were transferred to another plate. Leukocyte-sensitized Caco-2 cells continued to produce a high level of TNF-ot and to a lesser extent, IL-lB. They concluded that cross talk between Caco-2 cells and underlying immune cells is necessary for Caco-2 cells to recognize and respond to non- pathogenic bacteria. It was hypothesized that differences between fermented and non-fermented milk could stimulate different amounts of IL-6 from Caco-2 cells. Fermented cultures induced significantly more IL-6 than non-fermented cultures at 24, but not at 48 h. In the process of yogurt production, fermentation by LAB and bifidobacteria changes the composition of milk. Yogurt has increased folic acid, lactic acid and decreased lactose and vitamin B5 compared to non-fermented milk (Meydani and Ha, 2000; Shahani and Chandan, 1979). Calcium is also more bioavailable from yogurt. Bacterial enzymes can break down proteins and lipids in milk. It is possible that the digestion of certain milk proteins could result in the production of bioactive milk peptides that may have immunomodulatory activity (McDonald and others, 1994; Laffineur and others, 1996). The inactivation carried out in this research may have created compounds with suppressive effects from the bioactive milk peptides or substances associated with the fermentation process. The mode of bacterial inactivation did have a significant effect on IL-6 production. The main difference between these two modes of inactivation is that heat denatures proteins in the milk as well as on the surface and cytoplasm of the probiotic bacteria. In contrast, irradiation causes molecular changes in the DNA leaving the protein structure intact. These changes eventually lead to alterations in metabolism that can result in cell death if the irradiation damage is sufficiently extensive (Olson, 1998). Heat treatment of the cultures resulted in greater amounts of IL-6 compared to the NFDM control (Figures 4.2 and 4.3). The irradiated culture, however, resulted in lower amounts of IL-6 compared to the irradiated NFDM control. Many previous in vitro studies used heat-killed probiotic cultures and demonstrated immunostimulatory capabilities of the cultures. Comparison between the two modes of inactivation, heat vs irradiation, sought to determine if heat-denatured proteins from the cultures were responsible for cytokine stimulation as seen in previous studies (Marin and others, 1998; Park and others, 1999). Lysis of the bacterial cell is also possible with heat inactivation. The release of proteases, DNA, cytoplasmic proteins and exposure of new epitOpes could also be responsible for cytokine stimulation. 65 Although the stimulation and suppression of IL-6 production by heat-killed and irradiated cultures, respectively, were not significantly different from the naive Caco-2 supernatant, our results suggest that heat inactivation leads to the generation of stimulatory factors on the cultures. These stimulatory factors may be recognized by membrane bound Toll-like receptors (TLR) on the Caco-2 cells, that can recognize microbial components (Matzinger, 2002). TLRs can induce an immune response, including the production of cytokines. Perhaps the differences between heat and irradiation inactivation would be more pronounced using the co-culture system of Haller and others (2000). The current definition for probiotics stipulates that they should be ingested live to have immunostimulating effects in the body. While live bacteria were not investigated here, this research suggests that inactivated probiotics, specifically by irradiation, can suppress IL-6 production by gut epithelial cells. Because IL-6 plays a role in many immune functions, as well as inflammatory responses, its suppression may or may not be desirable. 4.2 Effect of lactic acid bacteria and bifidobacteria on lL-8 production by Caco-2 cells Heat-killed and irradiated lactic acid bacteria and bifidobacteria were incubated with a monolayer of Caco-2 cells for 24 and 48 h. The supernatants were collected and then analyzed for the cytokines IL-6 and 1L-8 using ELISA. Due to the differences in baseline cytokine production between replications, percent change in cytokine production was calculated where the amount of cytokine in the Caco-2 DMEM supernatant alone was 100%. When stimulated by IL-lB, the positive control, Caco-2 cells produced 11.6 to 90 times more IL-8. Uninoculated, reconstituted NFDM alone served as a negative control and did not significantly affect IL-6 and IL-8 production by Caco-2 cells during the 24 and 48 h incubations. The ‘PROC MIXED’ statistical procedure was used to determine the significance of each main effect interaction (i.e. culture by fermentation, culture by method of inactivation). The data were organized so that two sets of comparisons could be made. The first set accounted for all non—fermented samples at 10°, 107, and 10° CFU/ml, with the exception of ST133 which could not be grown to 10° CFU/n11. A separate analysis that did include ST133 indicated that it was not significantly different (p>0.05) fiom the other probiotic organisms at concentrations of 10° and 107 CFU/ml for any of the effects tested. The second set of data compared all fermented and non-fermented samples at 10° anle7 CFU/ml. Standard errors of the means were not included in the figures because they were generated based on the square root of the data. This was done to normalize the data. 4.2.1 Effect of culture The type of culture was only significant (p<0.05) to 1L-8 production when comparing the non-fermented cultures at 24 h (Table 4.5). LC stimulated significantly greater IL-8 (116.9%) (p<0.05) than NFDM (68%). None of the cultures, however, caused IL-8 production which was significantly different than levels of IL-8 in the Caco- 2 supernatant. At 48 11, the type of culture was no longer significant (p>0.05) (Table 4.6). 67 Table 4.5 ANOVA table comparing non-fermented cultures at concentrations of 10°, 10‘, and 10’ CFU/ml on ma production by Caco-2 cells after 24 hr incubation Effect Degrees of F Value Pr > F Freedom Culture‘ 250 2.26 0.0299 Concentration2 713 1.15 0.3176 Inactivation° 620 12.6 0.0004 Culture“ Concentration 707 1 .91 0.0266 Culture*Inactivation 226 2.66 0.0116 Inactivation*Concentration 713 0. 12 0.8878 ‘ Probiotic cultures 210", 102, 10“ CFU/ml 2 Heat-killed/Irradiated 68 Table 4.6 ANOVA table comparing non-fermented cultures at concentrations of 10°, 107, and 10° CFU/ml on IL-8 p_roduction llCaco-Z cells after 48 hr incubation Effect Degrees of F Value Pr > F Freedom Culture‘ 621 1.62 0.1267 Concentration2 708 2.04 0.1302 Inactivation3 712 2.85 0.0920 Culture‘Concentration 708 6.21 <0.0001 Culture*1nactivation 610 7.90 <0.0001 Irlactivation*Concentration 708 2.35 0.0959 m l Probiotic cultures 210“, 102, 108 CFU/ml 2 Heat-killed/Irradiated 69 Table 4.7 ANOVA table comparing fermented and non-fermented cultures at concentrations of 10° and 107 CFU/ml on lL-8 production by Caco-2 cells after a 24 hr incubation Effect Degrees of F Value Pr > F Freedom Culture 901 1.04 0.3988 Fermentation 992 O. 13 0.721 1 Concentration 1009 0.45 0.5013 Inactivation 1016 39.1 1 <0.0001 Culture‘Fermentation 884 0.64 0.7247 Culture*Concentration 1010 1 .38 0.2088 Culture‘lnactivation 934 3.36 0.0015 Fermentation*Concentration 1009 7.99 0.0048 Fermentation*lnactivation 910 0.47 0.4921 Inactivation“Concentration 1009 0.44 0.508 1 l Probiotic cultures 2 F ermented/Non-fermented 3 10", 107 CFU/ml 4 Heat-killed/Irradiated 70 Table 4.8 ANOVA table comparing fermented and non-fermented cultures at concentrations of 10° and 107 CFU/ml on IL-8 production by Caco-2 cells after a 48 hr incubation Effect Degrees of F Value Pr > F Freedom Culture 961 0.70 0.6748 Fermentation 1004 0.23 0.6314 Concentration 1003 8.85 0.0030 Inactivation 1010 18.33 <0.0001 Culture*Fermentation 948 3.41 0.0013 Culture*Concentration 1 004 2. 94 0.0047 Culture*Inactivation 977 2.77 0.0074 Fennentation*Concentration 1003 0. 1 6 0.6847 Fermentation‘lnactivation 971 1.00 0.3185 Inactivation*Concentration 1003 l .57 0.2 104 ; Probiotic cultures 3 Ferémenlted/Non-fermented 4 10 , 10 CFU/ml Heat-killed/Irradiated 71 In a comparison of fermented and non-fermented samples, the effect of culture was not significant (p>0.05) to IL-8 production at either time point (Tables 4.7, 4.8). With the exception of the difference between NFDM and LC at 24 h in the comparison of the unfermented cultures, the results indicate that culture does not have a significant effect on IL-8 production by Caco-2 cells. 4.2.2 Effect of @se Dose was significant when the IL-8 production by fermented and non-fermented cultures was evaluated at 48 h (Table 4.8). Cultures at a concentration of 10° CFU/ml significantly suppressed (p<0.05) IL-8 (75.4%) compared to cultures at 107 CFU/ml (83.4%). This comparison indicates that as the concentration of the culture increased, the amount of IL-8 induced by Caco-2 cells also increased. 4.2.3 Effect of fermentaLion There were no significant differences (p>0.05) between fermented and non- ferrnented cultures after 24 or 48 h of incubation (Tables 4.7 and 4.8). 4.2.4 Effect of inactivation A comparison of the non-fermented cultures determined that the mode of inactivation was significant at 24 h (p<0.05) (Table 4.5), but not at 48 h (Table 4.6). At 24 h, heat-killed cultures stimulated IL-8 (106.9%), whereas irradiated cultures suppressed IL-8 production (84.8%). 72 When fermented and non-fermented cultures were evaluated together, the mode of inactivation was significant at 24 and 48 h (p<0.05). After 24 h of incubation, heat- killed samples stimulated IL-8 production 108.7%, whereas irradiated samples suppressed IL-8 production 78.2%. At 48 h, the amounts of IL-8 decreased where heat- killed samples resulted in 85.3% IL-8 and irradiated cultures suppressed 1L-8 even further to 73.6%. The amount of IL-8 may have decreased at 48 h because the cytokine degraded or denatured over time. These results indicate that the mode of inactivation is an important factor of experimental design and should be carefully considered in future experiments. 4.2.5 Effect of the interaction between culture and dose The interaction of culture and dose was significant when comparing the non- fermented cultures at 24 (Table 4.5) and 48 h (Table 4.6). The individual comparisons that were significant (p<0.05) at 24 11, however, were between different cultures. We were only interested in different doses of the same culture and therefore these comparisons were not valid for this study. At 48 h, LB at a concentration of 10° CFU/ml significantly suppressed lL-8 production compared to LB at concentrations of 10° and 107 CFU/ml (Figure 4.5). This trend is opposite fiom several other studies, where it was observed that the higher the dose, the greater the cytokine response (Marin and others, 1998) When comparing the fermented and non-fermented cultures, the interaction between culture and dose was significant at 48 (Table 4.8) but not 24 h (Table 4.7). Caco-2 cells may have needed the longer incubation with the cultures in NFDM to 73 120 a 100 TV 6 f.;.-‘ [LL] :1. , o .3 a .3 80 - _: o . E. 1M ‘2? 2732316 ..... 13",: a 60 '1 ' 1.3:. .E 7i . & g 40 '1 b D °\° . . 20 4 , 0 1 l ' ‘ ' T 10" 10" 108 CFU/n11 Figure 4.5 Effect of dose of non-fermented heat- and irradiation-inactivated LB on IL-8 production by Caco-2 cells (48 h incubation). Unstimulated IL-8 in Caco-2 supernatant was considered 100%. ”b Concentrations with different letters are significantly different fi'om each other (p<0.05). 74 - 106 CFU/ml :1 107 CFU/ml 100 40-1 % Change in IL-8 production mv—r-y v.'—rT‘TYTAI v—rvr—r—r—.v—v I“? .rr r. Ver-‘r...| VV 1.. n ‘ II or! ..vr. ... .. . ._,.,. . ._ ,.... .........1 ........-.-»....1r-..- ...... .- . M.... .....v.. ....4.......1.. ,..........................., ..... 20-1 .. ‘ *e' 1?: it; o l 1 ° 1 ' ° ° ' I“ NFDM LA L8 L0 LR ST133 M1014 are Culture Figure 4.6 Effect of concentration (10° and 107 CFU/ml) of fermented and non- fermented lactic acid bacteria and bifidobacteria on IL-8 production by Caco-2 cells (48 h incubation). Unstimulated IL-8 in Caco-2 supernatant was considered 100 percent. * Indicates significant difference between concentrations 106 and 107 CFU/n11 for that culture (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgarr‘cus NCK 231; LC = L. casei ATCC 35935; ST133 = S. thermophilus Stl33; M1014 = B. adolescentis M101-4; BF6 = Brfidobacterium Bf-6. 75 produce IL-8 which demonstrated this interaction. With the exception of LB and LR, cultures at 107 CFU/ml resulted in greater IL-8 production than cultures at 10° CFU/ml (Figure 4.6). Only LA, however, demonstrated a significant difference (p<0.05) between the two concentrations. All of the cultures, including NFDM, suppressed IL-8 production relative to the Caco-2 supernatant. There may be a substance in the NFDM that caused this effect on IL-8 production. 4.2.6 Effect of the W The interaction of culture and mode of inactivation was significant (p<0.05) for comparisons of non-fermented cultures alone at both time points. At 24 h (Table 4.5), however, the specific effects of non-fermented cultures and mode of inactivation that were significant were between different cultures, not the same culture. Therefore, these results were disregarded. At 48 h (Table 4.5), heat-killed LB and LC cultures stimulated significantly higher amounts of IL-8 fi'om Caco-2 cells than their irradiated counterparts (Figure 4.7). The irradiated cultures of BF6, LB, and LC significantly suppressed IL-8 production compared to the Caco-2 supernatant. Interaction of culture and inactivation was also significant (p<0.05) for comparisons of fermented and non-fermented cultures at 24 (Table 4.7) and 48 h (Table 1 4.8). At 24 11, the heat-killed samples of NFDM and BF6 stimulated significantly greater (p<0.05) amounts of IL-8 than their irradiated counterparts (Figure 4.8). Although not significant (p>0.05), the trend of heat-killed culture resulting in greater levels of IL-8 than the irradiated culture was seen for all cultures tested. It could be that irradiation resulted in some kind of change in the NFDM that was inhibitory to IL-8 production. 76 120 -Heat-killed -lrradiated * 100 , _ «'3’ 2°‘ 8 :51 3 ct. 1 °9 1325 :33 .. =1 3°15: ,g :63 a: .1: ii- :2; 1 8) r .. . .. .. .11 g 40‘ 9} U . o5 20- 1 :1 1 . 0 :1 E l NFDM LA LB LC LR ST133 M1014 BF6 Culture Figure 4.7 Effect heat and irradiation inactivated non-fermented lactic acid bacteria and bifidobacteria at concentrations of 10°, 107, and 10° CFU/ml on IL-8 production by Caco-2 cells (48 h incubation). Unstimulated IL-8 in Caco-2 supernatant was considered 100%. * Indicates that heat-killed were significantly difl‘erent than irradiated counterpart (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgaricus NCK 231; LC = L. casei ATCC 39539; LR = L. reuterr‘ ATCC 23272; ST133 = S. thermophilus Stl33; M1014 = B. adolescentis M101-4; BF 6 = Brfidobacterium Bf-6 77 140 . - Heat-killed * 120 - Irradiated 100 __ op P: d 80 - .. i F :33 '5 ‘ ° ”T m 1- E 60 7 5; U 53’? 1‘5? ‘1-i °\° 1 40 7 3? 1 .1 20 1 *1 52:: i. .. 9; 1 :.£ ‘ 0 .. I , I . t" 1 NFDM LA LB LC LR ST133 M1014 BF6 Culture Figure 4.8 Effect of heat and irradiation inactivation on fermented and non- fennented lactic acid bacteria and bifidobacteria at concentrations of 10° and 107 CFU/ml on IL-8 production by Caco-2 cells (24 h incubation). Unstimulated IL-8 in Caco-2 supernatant was considered 100 percent. * Indicates that heat-killed was significantly different than irradiated counterpart (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgaricus NCK 231; LC = L. casei ATCC 39539; LR = L. reuterr’ ATCC 23272; ST133 = S. thermophilus Stl33; B. adolescentis M101-4; BF6 = Bifidobacteriwn Bf-6. 78 The irradiated sample of NFDM alone suppressed IL-8 production the most and was significantly different than the level of IL-8 in the Caco-2 supernatant. After the 48 h incubation, the interaction of culture and mode of inactivation for fermented and non-fermented cultures was still significant (p<0.05) (Table 4.8). More specifically, heat-killed LB cultures suppressed IL-8 (95.5%), which was significantly more IL-8 than the suppression by irradiated LB cultures (65.4%) (Figure 4.9). As with the 24 h cultures, most irradiated cultures at 48 h suppressed lL-8 production more than the heat-killed cultures. 4.2.7 Effect of the interaction between culture__ and fermentation Although the interaction of culture and fermentation was found to be statistically significant at 48 h (Table 4.8), the specific interactions were between different cultures not among the same culture. These interactions were not valid for this study. 4.2.8 Effect of the interaction between fermentation and concermtion The interaction between fermentation and concentration was found to be significant at 24 h (p<0.05). Upon closer examination, however, there were no individual differences. This can occur in multiple comparison statistical analysis when the sample sizes are not balanced (Smith, 2002) 79 - Heat-killed Irradiated 100 c. 80 1 i g " i" “:3, j 1.— " r' 5— 5° * 1 ‘ *-:j 0° 3:: f? 3% d. 5;: 12'; :3 i: o _ :32 :i 3?: -= 1* {i1 5“ U :1 :5 5; o\° if: s5 it ‘3 5:11 62.; if? 135 2:: 3:1 11‘ o r .. . '1 . ‘. . I . I , I .. NFDM LA LB LC LR ST133 M101 BF6 Culture Figure 4.9 Comparison of heat and irradiation inactivation of fermented and non-fermented lactic acid bacteria and bifidobacteria at concentrations of 10‘5 and 107 CFU/ml on IL-8 production by Caco-2 cells (48 h incubation). Unstimulated IL-8 in Caco-2 cell supernatant was considered 100%. * Indicates heat-killed were significantly different from irradiated counterpart (p<0.05). NFDM = non-fat dry milk; LA = L. acidophilus LA2; LB = L. bulgaricus NCK; LC = L. casei ATCC 39539; LR = L. reuteri ATCC 23272; ST133 = S. thermophilus Stl33; M1014 = B. adolscentis MIDI-4; BF6 = Bifidobacterium Bf96 80 4.2.9 Discujssjon on th_e_efl‘ect MW production by Caco-2 cells While some significant differences were noted between LC and NFDM, there were no consistent patterns to the effect of culture on IL-8 production. As was true for IL-6, none of the cultures significantly stimulated (p>0.05) IL-8 production at any dose which was contrary to experiments conducted by Marin and others (1998) who examined the effect of LAB and bifidobacteria on cytokine production by mouse RAW 264.7 macrophage cells and mouse EL4.IL-2 thymoma cells. All bacteria were heat-killed and incubated with cell lines at concentrations of 106, 107, and 108 bacteria/ml. Probiotic strain- and dose-dependent increases were observed with respect to IL-6 and TNF-a production by RAW 264.7 cells as well as in IL-2 and IL-5 production by EL4.IL-2 cells. Compared to other bacteria studied, S. thermophilus ST133 (also used in our study) had the greatest enhancing effects on cytokine production. In general, they observed that as the concentration of all bacteria increased, so did the amount of cytokine produced by the respective cell lines. The effect of dose was only significant when comparing fermented and non- fermented cultures, but not for non-fermented cultures alone. Although the effect of fermentation alone was not significant, these results suggest that fermentation enhances the effect of dose. It was hypothesized that differences between fermented and non- fermented milk could stimulate different amounts of IL-8 from Caco-2 cells. During yogurt production, fermentation by LAB and bifidobacteria changes the composition of milk. Yogurt has increased folic acid, lactic acid and decreased lactose and vitamin B6 compared to non-fermented milk (Meydani and Ha, 2000; Shahani and Chandan, 1979). 81 Calcium is also more bioavailable from yogurt. Bacterial enzymes can break down proteins and lipids in milk. It is possible that the digestion of certain milk proteins could result in the production of bioactive milk peptides which may have immunomodulatory activity (McDonald and others, 1994; Laffineur and others, 1996). The inactivation carried out in this research may have created compounds with suppressive effects fiom the bioactive milk peptides or substances associated with the fermentation process. There are other reports of strain and concentration effects of probiotics and their components on cytokine production (Park and others, 1999; Tejada-Simon and Pestka, 1999; Miettinen and others, 1996). Our results were contrary to these previous studies, possibly because the cell models differed. It is also possible that Caco-2 cells require communication with underlying immune cells in order to respond to Gram-positive bacteria. Haller and others (2000), as mentioned in section 2.7, used Caco-2 cells in a co- culture system with human blood leukocytes where the two types of cells were separated by a membrane in transwell culture plates. Without the leukocytes, Caco-2 cells could not be stimulated by L. sakei to produce cytokines TNF-or and IL-lB. After the initial incubation the two wells were separated. Leukocyte-sensitized Caco-2 cells continued to produce a high level of TNF-or and to a lesser extent, lL-IB. They concluded that cross talk between Caco-2 cells and underlying immune cells is necessary for Caco-2 cells to recognize and respond to non-pathogenic bacteria. The mode of bacterial inactivation did have a significant effect on IL-8 production. The main difference between these two modes of inactivation is that heat denatures proteins in the milk as well as on the surface of the probiotic bacteria. In contrast, irradiation leaves the protein structure intact, but causes molecular changes in 82 the DNA. These changes eventually lead to alterations in metabolism which can result in cell death if the irradiation damage is sufficiently extensive (Olson, 1998). Heat-treated cultures generally resulted in greater amounts of IL-8 compared to the irradiated cultures. These amounts of IL-8, however, were lower than the Caco-Z supernatant and were unexpected based on previous experiments which used heat- inactivated cultures (Marin and others, 1998; Park and others, 1999). The comparison between the two modes of inactivation, heat vs irradiation, in our experiments sought to determine if heat-denatured proteins on the cell surface of the cultures were responsible for cytokine stimulation as seen in previous studies. Although the stimulation or suppression of IL-8 production by heat-killed and irradiated cultures, respectively, in our experiment were not significantly different from the naive Caco-2 supernatant, these results suggest that heat inactivation leads to the generation of stimulatory factors on the cultures. These stimulatory factors may be recognized by membrane bound TLR on the Caco-2 cells, that can recognize microbial components (Matzinger, 2002). Perhaps the differences between heat and irradiation inactivation would be more pronounced using the co-culture system of Haller and others (2000) The current definition for probiotics stipulates that they should be ingested live to have immunostimulating effects in the body. While live bacteria were not investigated here, this research suggests that inactivated probiotics, specifically by irradiation, can suppress IL-8 production by gut epithelial cells. Because IL~8 plays a role in many immune fiJnctions, particularly in the inflammatory response, its suppression may or may not be desirable. 83 4.3 Future research for lactic acid bacteria and bifidobacteria in NFDM on cytokine production by Caco—2 cells Future research could utilize the co-culture system previously described to determine if the probiotic cultures used in these experiments are capable of stimulating cytokine production in Caco-2 cells. Experiments with the co-culture system should investigate a dose effect of probiotic bacteria in NFDM on cytokine stimulation by human Caco-2 cells. Based on the previous in vitro studies on probiotic dose, the probiotic organisms used in these experiments would likely cause cytokine production by Caco-2 cells with the aid of underlying immune cells. 4.4 Effect of milk components on cytokine production by Caco-2 cells Although not statistically significant, a trend was observed fi'om the previous experiments where NFDM suppressed IL-6 and IL—8 production by Caco-2 cells. Thus, the intent of the next experiment was to investigate the effect of individual milk components on IL-6 and IL-8. The milk components lactose, or-la, B-lg as well as NFDM were incubated with Caco-2 cells with or without IL-IB for 24 h. IL-6 and IL-8 production were calculated using the amount of cytokine in the Caco-Z DMEM supernatant alone as 100%. The milk component effect on IL-6 production by Caco-2 cells was statistically significantly (p<0.0001). Figure 4.10 illustrates IL-6 production by the specific milk components with and without IL-l B. The error bars represent standard error based on raw data. All milk components and NFDM stimulated significantly greater (p<0.05) IL-6 moo 1 M T 800 n, b ' G .2 ‘6 3 1: 95. 600 - c ‘9 C c c: 1:1 :2: .... 1; 3’0 40° 1 11.1; 1:; g a a a 1 :1 °\° ::: : 1T i : 1 ' 13 200 n 1 15,11 :11 .1.1,f1.1-1 .1.) ...... 1T w 1 ~ 7 ' 1 O f r "'1” T '1 i l I r T 1 V“ s? \V \V \V \r go it” 01,1: of we 041 31> Milk component Figure 4.10 Effect of various milk components on IL-6 production by Caco-2 cells with or without stimulation by IL-lbeta. NFDM= non-fat dry milk, IL=IL-1beta; alpha-LA = alpha-lactalbumin; beta-LG = beta-lactoglobulin. IL-6 produced by Caco-Z supernatant alone was considered 100 percent IL-6 production. "d Indicates that means with different letters are significantly different from each other (p<0.05). 85 than was present in the naive Caco-2 cell supernatant. IL-6 production stimulated by lactose and a-la were not significantly different from NFDM or each other. Beta-lg alone, however, stimulated significantly greater IL-6 production than NFDM and caused Caco-2 cells to produce 726% more IL-6 than the Caco-2 supernatant. This was significantly more (p<0.05) IL-6 than the samples with IL-IB alone, NFDM with lL-IB, lactose with IL-IB and a—la with IL—IB. Although [Hg with IL-lB stimulated the most IL-6 (931.4%) ofall milk component samples, it was not significantly greater than the [3- lg alone. The effect of milk components on IL-8 production by Caco-2 cells was also significant (p<0.0001), but did not follow the same trends as for IL-6 production. Figure 4.11 shows the effect of the specific milk components on IL-8 production by Caco-2 cells. The error bars represent standard error based on raw data. All milk components, with the exception of NFDM and lactose, stimulated significantly greater (p<0.05) IL-8 than the naive Caco-2 cell supernatant. Alpha-la and [Hg stimulated significantly (p<0.05) more IL-8 than NFDM and lactose. Although not significant, a-la caused Caco-2 cells to produce more (596.2%) 1L-8 than IL-lB (468.1%) (p>0.05). The combination of milk components and NFDM with IL-lB had an additive effect on IL-8 production (p<0.05). Several milk components have been reported to have immunostimulating effects (Gill and others, 2000). In our study, IL-6 stimulation by all milk components was at least 200% greater than the Caco-2 supernatant. Beta-lg is considered a potent milk allergen (Tsuji and others, 200]). Jenmalm and others (1999) found that B—lg could 86 % Change in IL-8 production 1200 - 1000 l I try... ”#553951 M52155} 400 { 73-73 T. 200 J fcf$f off Milk components Figure 4.1] Efi‘ect of various milk components on IL-8 production by Caco-2 cells with or without stimulation by IL-lbeta. NFDM = non-fat dry milk; alpha-LA = alpha-lactalbumin; beta-LG = beta- lactoglobulin; IL = IL-lbeta. IL-8 in Caco-Z supernatant was considered 100 percent IL-8 production. "° Indicates that means with different letters are significantly different from each other (p<0.05). 87 induce increased lL-6 by PBMCs from eight-year old children with and without atopic symptoms. Afier 96 h of incubation with B-lg, PBMCs from atopic children produced as much as 100 ng/ml IL-6, which was significantly greater (p<0.05) lL-6 than those from children without atopic symptoms at 60 ng/ml. While it is a different cell system, their results may be an indicator of the stimulatory effect of B-lg in the body. Comparing the milk components and NFDM without IL-IB, a-la was a more potent stimulator of IL-8 than even IL-IB alone. Wong and others (1997) found that a- LA (400 ug/ml) increased IL-lB production by ovine blood lymphocytes 56.9 percent. Although the cell models are different, it is possible that a-la could increase IL-IB production by Caco-2 cells and as a result, increase IL-8 production. These experiments investigating cytokine production by milk components gave conflicting results compared to the previous two experiments. Previously, NFDM suppressed cytokine production. In these experiments, NFDM and the milk components stimulated IL-6 production and a-la and B-lg stimulated IL-8 production. This could be due to the fact that the milk components and NFDM solutions were more concentrated (4% solution in culture) than in experiments with the probiotic organisms (2% NFDM solution in culture). The purity of the milk components also could have played a role in the results. According to Wong and others (1997), the more pure bovine whey fractions were, the more clear-cut the immune response observed. Further experiments with dose of NFDM and milk components should be conducted to detemline the effects on cytokine production by Caco-2 cells. 88 4.4.1 Future rem Because ot-la and B-lb in 4% solutions stimulated IL-6 and IL-8 production, experiments with dose of these and other milk components should be performed to understand their role in cytokine production by Caco-2 cells. Irradiation of the milk component solutions would also help to understand the differences seen between heat- and irradiation-inactivated samples in the previous experiments with probiotic cultures. Perhaps a co-culture system would give further insight as to the function of milk components in the gut. 89 CHAPTER 5 SUMlVIARY CHAPTER 5 SUMMARY In this research, we conducted experiments to determine the immunomodulatory effects of dose and inactivation of seven different probiotic cultures on the human Caco-2 cell culture system. Further experiments with milk components and the Caco-Z cell line were also conducted to determine if any of the components had an inhibitory effect on immune responses. The results of the investigation with probiotics suggest that Caco-2 cells were largely unresponsive to incubation with the cultures. The majority of the cultures did not significantly suppress or stimulate cytokine production (IL-6 and IL-8) (p>0.05). The dose of culture also did not have a significant effect. Although it was not significant for every culture, the mode of culture inactivation had an effect on cytokine production. In general, IL-6 and IL-8 production by Caco-2 cells were suppressed by irradiated probiotic cultures more than their heat-killed counterparts. We also discovered that the soluble whey proteins, a-la and B-lb, stimulated more cytokine production than the other milk components examined. 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Dose rates were measured with Renter-Stokes ion chamber model RS-C4-1606-207, serial number Z-8943, which is calibrated annually by the manufacturer or Phoenix against a National Institute of Standards 8: Technology source. Organization: H 5 U " [and finance} “Mm “an IV- 95') Irradiation Date: 9/20/0 I Specimen'l‘ype: Ac' 4’ r' ' la. e Specimen Identification: 05 2629/ Hit/“F59; ‘ Disrance from Irradiator (cm): _C&I_ ML Gamma Dose Rate (rad/hr): @8850, , Irradiation Time (hr): 0,8! 7 (firazlaév‘i'ms) Interrupt Time (min): J Gamma Dose (Mrad): /- 00 Amp 20, zeal WW / Date ’ Robert B. Blackburn Asst. Manager of laboratory Operations 104 APPENDIX 11 105 MICHIGAN STATE UNIVERSITY March 9. 2000 TO: Zeynep USTUNOL 136 GM. Trout Food Science Bldg RE: IRB# oo-120 CATEGORYz1-E APPROVAL DATE: March 7 2000 TITLE. EFFECT OF LACTIC ACID BACTERIA AZND BIFIDO-BACTERIA ON THE CYTOKINE PRODUCTION BY CACO-2 CELLS The University Committee on Research Involving Human Subjects' (UCRII-IS) review of this project is complete and I am pleased to advise that the rights and welfare of the human subjects appear to be adequately protected and methods to obtain informed consent are appropriate. Therefore. the UCRIHS approved this project. RENEWALS: UCRIHS approval is valid for one calendar year, beginning with the approval date shown above. Projects continuing beyond one year must be renewed with the green renewal form. A maximum of four such expedited renewals possible. Investigators wishing to continue a project beyond that time need to submit it again for a complete review. REVISIONS: UCth-IS must review any changes in procedures involving human subjects. prior to initiation of the change. If this is done at the time of renewal. please use the green renewal form. To revise an approved protocol at any other time during the year. send your written request to the UCRIHS Chair. requesting revised approval and referencing the project' s "281! and title. Include in your request a description of the change and any revised instruments consent forms or advertisements that are applicable. PROBLEMS/CHANGES: Should either of the following arise during the course of the work. notin UCRIHS promptly: 1) problems (unexpected side effects. complaints. etc.) involving human subjects or 2) changes in the research environment or new information indicating greater risk to the human subjects than existed when the protocol was previously reviewed and approved. If we can be of further assistance, please contact us at 517 355-2180 or via email: UCRIHSQpiIoLmsuedu. Please note that all UCRIHS forms are located on the web: http:/Mww.msu.eduluserlucrihsl Sincerely. cc: Constance Wong 2125 8. Anthony . 106 MICHIGAN STATE u N l v r: R s r T Y Febmary?8.2001 TO: Zeynep USTUNOL 2105 3. Anthony Hall RE: IRB# 00-120 CATEGORY: EXEMPT 1-E RENEWAL APPROVAL DATE. February 27, 2001 TITLE: EFFECT OF LACTIC ACID BACTERIA AND BIFIDO—BACTERIA ON THE CYTOKINE PRODUCTION BY CACO-Z CELLS The University Committee on Research Involving Human Subjects' (UCRIHS) review of this project is complete and I am pleased to advise that the rights and welfare of the human subjects appear to be adequately protected and methods to obtain informed consent are appropriate. Therefore. the UCRIHS APPROVED THIS PROJECTS RENEWAL RENEWALS: UCRIHS approval is valid for one calendar year. beginning with the approval date shown above. Projects continuing beyond one year must be renewed with the green renewal form. A maximum of four such expedited renewal are possible. Investigators wishing to continue a project beyond that time need to submit it again for complete review. REVISIONS: UCRIHS must review any changes in procedures involving human subjects. prior to initiation of the change. If this is done at the time of renewal. please use the green renewal form. To revise an approved protocol at any other time during the year. send your written request to the UCRIHS Chair. requesting revised approval and referencing the project‘s IRB# and title. Include in your request a description of the change and any revised instruments. consent forms or advertisements that are applicable. PROBLEMS/CHANGES: Should either of the following arise during the course of the work. notify UCRIHS promptly: 1) problems (unexpected side effects, complaints. etc.) involving human subjects or 2) changes in the research environment or new information indicating greater risk to the human subjects than existed when the protocol was previously reviewed and approved. If we can be of further assistance. please contact us at 517 355-2180 or via email: UCRIHS@pilot.msu.edu. Ashir Kumar M. D. Interim Chair UCRIHS AK: 13' cc: Constance Wong .2125 S. Anthony 107 MICHIGAN STATE u N l v E R s I T Y January 31. 2002 TO: Zeynep USTUNOL 2105 S. Anthony Hall MSU RE: IRBI 00-120 CATEGORY: 1-E EXEMPT RENEWAL APPROVAL DATE: Januay 30, 2002 TITLE: EFFECT OF LACTIC ACID BACTERIA AND BlFlDO-BACTERIA ON THE CYTOKINE - PRODUCTION BY CACO-2 CELLS TheUniversityCommitteeonReswch InvolvingHumanSubjects'(UCRII-IS)reviewofthisproject BeanpleteaMlamueasedbadviseumtmenghtswvelmdflnmwbjecbappeab beadequatelyprotectedandmethodstoobtain informedconsentareappropriate. Thereforethe UCRIHS APPROVED THIS PROJECTS RENEWAL RENEWALS: UCRIHS approval is valid for one calendar year. beginning with the approval date shownabove. ProjectsconfimmgbeymdomyurmudbermewedmmegremmfonmA maximum offousuchexpeditedrenewatarepossible. lnvestigatorswishingtocontiueapmject beyondthattimeneedtosubmititagainfor completereview. REVISIONS: UCRIHSmustreviananydiangesmprocedueshvdvmglumansubjects.uiato hitiationofthechange. flfliisisdmeatmefimeofrenewat.pleaseusemegreenrenewalfmn.To revisemapwwedwdocdatanyoflrafimedmhgflnyea.smdmwihenrequestbm UCRIHSChai.mqueefingrevisedapumdmdrefaanhgdnprojed‘isB#aMfifle. Includein yoummrestadesaipfiondflndrmgemdanyrevisedhstuments,camflfamsu advertisementsthatareapplimble. - PROBLEMSI‘CI-IANGES: Shouldeithercfthefcnawingariseduingthecomseofthemm UCRIHS promptly: 1)problems(unexpectedsideeffects. complaints. etc.)invclvhgtunansubjects «2)dimgeshwreseadimnauananiuamafimmdhafingg'atariskmmm wbjedsmaneidstedmmeprotccdwasprevbuslyreviemdandappmved. NwecenbeofWassistance.pIeasecontadusat517355—2180uviaemaiz UCRIHSQpioLmsuedu. 108 ll”. "II II IIIIIIIIIIIIIIII II" 1293 02334 289 6