Nghiên cứu Whey Protein và Cystic Fibrosis: Ảnh hưởng đến viêm nhiễm tại các tế bào biểu mô đường hô hấp

EFFECT OF NATIVE AND PRESSURIZED WHEY PROTEIN ISOLATES ON INFLAMMATION IN RESPIRATORY EPITHELIAL CELLS EXPRESSING EITHER WILDTYPE OR MUTANT CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) Michèle Michel Iskandar, M. School of Dietetics and Human Nutrition McGill University, Montreal Quebec, Canada June 2011 A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Doctor of Philosophy © Michèle M. Iskandar, 2011 This thesis is dedicated to my grandparents, Juliette, Romaine, Edmond, and particularly Georges, whose foresight made this possible. ii "Science is a wonderful thing if one does not have to earn one’s living at it.” Albert Einstein iii TABLE OF CONTENTS TITLE PAGE.

i TABLE OF CONTENTS. x ADVANCE OF SCHOLARLY KNOWLEDGE. xii CONTRIBUTIONS OF CO-AUTHORS TO MANUSCRIPTS. xv LIST OF TABLES.

xvii LIST OF FIGURES. xviii LIST OF ABBREVIATIONS.1 Rationale and Statement of Purpose.1 Cystic Fibrosis: Epidemiology and Pathophysiology.2 Cystic Fibrosis: Inflammation and Anti-inflammatory Therapy.3 Pathways to IL-8 Secretion.4 Oxidative Stress in CF.5 Therapies for CF.2 Whey Proteins: an Overview.1 Antioxidant Potential of Whey Proteins and Peptides.2 Immunomodulatory and Anti-Inflammatory Potential of Whey Proteins and Peptides.3 Protein Modification by Hyperbaric Pressure Treatment.4 Effect of High Pressure on Whey Proteins. “Effect of Hyperbaric Pressure on the Digestibility and Antioxidant Potential of Whey Protein Isolates”.2 Materials and Methods. “Inhibition of Interleukin-8 Release by Pressurized Whey Protein Hydrolysate in Respiratory Epithelial Cells: Mechanism of Action”.2 Materials and Methods.

“Dietary Supplementation with Pressurized Whey in Patients with Cystic Fibrosis”.2 Subjects and Methods. FINAL CONCLUSION AND SUMMARY. 177 v ABSTRACT Cystic fibrosis (CF) is a fatal inherited disease characterized by chronic, persistent and exaggerated inflammation. The airways of CF patients exhibit a sustained influx of neutrophils and interleukin (IL)-8, the major neutrophil chemoattractant.

Anti- inflammatory therapy is a focus of CF treatment, with a clear need for novel, safe and effective therapies. Whey proteins have been attributed a number of biological activities, including antioxidant and anti-inflammatory effects. Previous research has suggested a suppression of the inflammatory response to tumor necrosis factor (TNF)α by whey protein hydrolysates (WPH) in respiratory epithelial cell lines. Furthermore, it is suggested that hyperbaric pressure (HP) treatment of whey proteins can potentiate their biological activities.

The effect of HP treatment on the digestibility, anti-inflammatory and antioxidant activity of whey protein isolates (WPI) was explored in a series of mechanistic in vitro studies and in a clinical trial. Hydrolysates of pressurized (pWPH) and native (nWPH) whey protein isolates were generated using two differing in vitro digestion protocols that mimic human gastrointestinal digestion. Hyperbaric pressure pre- treatment resulted in enhanced in vitro digestibility and antioxidant activity of WPI regardless of major differences in enzymatic conditions and the resulting peptide profiles, signifying that pressure processing generates an enhanced release of bioactive peptides regardless of the proteolytic environment. The peptide profiles obtained from the hydrolysates of pWPH exhibited quantitative and qualitative differences from those of nWPH.

To explore the possible mechanisms by which WPH may exert their anti- inflammatory effects, wild-type and cystic fibrosis (CF) conductance regulator-deficient cells were treated with either nWPH or pWPH and stimulated with TNFα, interleukin (IL)-1β or lipopolysaccharide (LPS). Both nWPH and pWPH suppressed LPS-induced IL-8 secretion, although pWPH were more potent requiring lower doses to exert significant inhibition. pWPH increased the ferric-reducing antioxidant activity of cell culture supernatant in both cell lines. Neither type of hydrolysate suppressed TNFα- or IL-1β-induced IL-8 secretion.

Since LPS and IL-1β, beyond the activation of their respective receptors, share a common intracellular pathway, further experiments were conducted on signalling events that occur prior to the convergence of the two pathways, vi at the level of Toll-like receptor (TLR)4. There was no effect of either nWPH or pWPH on the cell membrane expression of TLR4. Neither type of WPH exhibited direct binding and neutralization of LPS, but both significantly reduced the binding of LPS to cell surface receptors. It is therefore likely that the WPHs exerted the observed LPS-induced IL-8 suppression by suppressing the binding of LPS to its receptor with the consequent inhibited activation of TLR4.

To explore the possible in vivo clinical effects of pressurized WPI, a one-month, open-label pilot study of supplementation with pressurized WPI in children and adults with CF was conducted. Nutritional status was enhanced in both children and adults. An improvement in lung function was observed in children, and C-reactive protein decreased in the majority of patients with initially high values. Overall, the thesis results provide a novel mechanism by which whey proteins and pressurized whey can exert anti-inflammatory effects and suggest new potential avenues for the use of pressurized whey protein isolate as a nutrition-based anti-inflammatory therapeutic or preventative agent.

vii RESUME La fibrose kystique (FK) est une maladie héréditaire fatale caractérisée par une inflammation chronique et exagérée. Les voies respiratoires des patients atteints de FK présentent un afflux soutenu de neutrophiles et d’interleukine (IL)-8, l’agent chimiotactique majeur des neutrophiles. La thérapie anti-inflammatoire est un focus du traitement de la FK et il existe un besoin évident de nouvelles thérapies sûres et efficaces. Des effets biologiques, incluant des effets antioxydants et anti-inflammatoires ont été attribués aux protéines de lactosérum.

Des recherches antérieures ont suggéré une suppression de la réponse inflammatoire induite par le facteur de nécrose tumorale (TNF)α par des hydrolysats de protéines de lactosérum (WPH). De plus, il a été suggéré que le traitement par pression hyperbare (PH) des isolats de protéines de lactosérum (WPI) peut potentialiser leurs activités biologiques. Les effets d’un traitement PH sur la digestibilité et les activités anti-inflammatoires et antioxydants des WPI ont été explorés dans une série d’études mécanistes in vitro et dans un essai clinique. Des hydrolysats de protéines de lactosérum pressurisées (pWPH) et non pressurisées (nWPH) ont été générés par le moyen de deux différents protocoles de digestion in vitro imitant le processus de digestion gastro-intestinal humain.

Le prétraitement HP a entrainé une augmentation de la digestibilité in vitro et de l’activité antioxydante des WPI, malgré les différences entre les deux protocoles de digestion et entre les profiles de peptides résultant des digestions, signifiant que la pression génère une augmentation des peptides bioactifs générés quel que soit l’environnement protéolytique. Les profiles des peptides des pWPH ont exhibé des différences qualitatives et quantitatives comparés aux nWPH. Pour explorer les mécanismes possibles par lesquels WPH pourraient exercer leurs effets anti- inflammatoires, des lignées cellulaires normales et déficientes du Régulateur de la Conductance Transmembranaire Mucoviscidose (CFTR) ont été traitées avec nWPH ou pWPH et stimulées avec TNFα, IL-1β, ou lipopolysaccharide (LPS). Les nWPH et pWPH ont diminué la sécrétion d’IL-8 induite par LPS, bien que les pWPH aient été plus puissants, requérant de moindres doses pour exercer une inhibition significative.

pWPH a augmenté l’activité antioxydante des surnageants des cultures des deux lignées cellulaires. Aucun type de WPH n’a inhibé la sécrétion d’IL-8 induite par TNFα ou IL- viii 1β. Puisque LPS et IL-1β partagent une voie commune de signalisation suivant l’activation de leurs récepteurs respectifs, des expériences ont été menées sur des événements prenant place en amont de la convergence des deux voies de signalisation, au niveau du récepteur Toll-like receptor (TLR)4. Les nWPH ni les pWPH n’ont eu d’effet sur l’expression du TLR4, mais la liaison entre LPS et les récepteurs de surface a été réduite par les deux traitements.

Aucun WPH n’a exhibé de liaison directe ou de neutralisation du LPS. Il est donc probable que les WPHs aient exercé la suppression d’IL-8 induite par LPS en inhibant la liaison du LPS avec son récepteur et par conséquent l’activation du TLR4. Pour explorer les effets cliniques in vivo des WPI pressurisés, un essai clinique pilote d’une durée d’un mois a été mené, où des enfants et adultes souffrant de FK ont été supplémentés avec des protéines pressurisées. Le statut nutritionnel des enfants et adultes a été amélioré.

Une amélioration des fonctions pulmonaires chez les enfants a été observée et le taux sanguin de protéine C réactive a diminué chez la majorité des patients chez qui les taux étaient élevés au début de l’étude. Globalement, les résultats de cette dissertation fournissent un nouveau mécanisme par lequel les WPI pressurisés peuvent exercer des effets anti-inflammatoires et suggèrent de nouvelles avenues potentielles pour l’usage des WPI pressurisés en tant qu’agent nutritionnel thérapeutique ou préventif. ix ACKNOWLEDGMENTS This work would not have been possible if it were not for many people who have supported me in many ways. My supervisors, Dr.

Larry Lands and Dr. Stan Kubow, have taught me that many things are possible in the pursuit of scientific knowledge with creativity, dedication, perseverance and positive thinking. I thank them both for their patience, their dedication to the work, and for sharing their extensive scientific experience with me. I would like to thank my committee member Dr.

Cameron Skinner, who welcomed me in his lab and generously took time to teach me and discuss the concepts of my thesis with me. I cannot underplay the help and encouragement I have received from Dr. He taught me everything I know about cell culture and was a constant presence throughout my work. He provided for lively scientific debates, took extra time to perform measurements in a timely fashion, and must have answered over a million of my questions over the years.

Thanks to Brian Meehan, who taught me basic skills when I knew so little, and gave me a curiosity to always look for more answers. I am very greatful to Lise Grant, who has always gone above and beyond to orient me within the system when I needed it, and always with a smile. Many thanks to Francine Tardif and Elizabeth Ansell for their professional support. Thanks to all the members of Dr.

Lands’ lab: Nadir Mawji, who reinforced my enjoyment of teaching and who was always eager to learn, and to help by any means he could. Kassey Herscovitch and Maria Camisano for their good nature, encouragement, and for sharing the burden of some experiments. Both Kassey and Maria provided much needed levity throughout stressful times. I thank the members of Dr.

Kubow’s lab: Andre Piccolomini who was always ready to help and his admirable positive attitude especially x under pressure, Kebba Sabally, Atef Nassar and Behnam Azadi for his good nature and for making himself available whenever he could. My friends who have always been positive and understanding: Maritza Laprize for her continued encouragement, Rima Itani, Lynn Khatib and Rima Awar who were present although miles away. Last, but not least I owe a debt of gratitude to my family; my father Michel and my mother Josee, my brother Eddy, for their great, unfailing and unconditional support and encouragement. Although they are hundreds of miles away, they are and have been an important source of comfort throughout every step of this process and of my life.

Without their love and support during difficult times, this work would not have been possible. I am greatful to my partner Alain, for becoming my family, my friend, and a tremendous, constant and loving presence by my side. xi ADVANCE OF SCHOLARLY KNOWLEDGE 1. Claims of Original Research In this thesis, it is demonstrated for the first time that: 1.

Hydrolysates from the in vitro digestion of whey protein isolates act upon wild- type and cystic fibrosis (CF) respiratory epithelial cells to decrease lipopolysaccharide (LPS)-induced interleukin (IL)-8 secretion. The mechanism by which whey protein hydrolysates interfere with LPS-induced IL-8 is through decreasing the binding of LPS to cell surface receptors. Treatment of whey protein isolates with hyperbaric pressure prior to in vitro digestion enhances the LPS-mediated interleukin-8 suppressing effects of the resulting hydrolysates. Treatment of whey protein isolates with hyperbaric pressure prior to in vitro digestion enhances the in vitro antioxidant capacity of the resulting hydrolysates in both aqueous cell-free and cell culture systems.

Short-term supplementation with pressurized whey protein improves BMI in adults, BMI z-score and lung function in children, and decreases serum C-reactive protein levels in patients with initially high values. This thesis presents improvements over previous research: 6.