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Heat Shock Protein 70 and the Relationship to Glutamine in the Critically Ill Child

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Heat Shock Protein 70 and the Relationship to Glutamine in the Critically Ill Child Heat Shock Protein 70 and the relationship to glutamine in the critically ill child Luise Marino BSc (Hons) Cat. & App. Nutrition, Post Grad Diploma Dietetics, MMed Sci Nutrition Paediatric Research Dietitian Section of Paediatrics Department of Infectious Diseases Faculty of Medicine Imperial College London St. Mary’s Campus Norfolk Place London Dedication To my angel, Zarah 2 Declaration In accordance with Imperial College London regulations, I declare that all work presented in this thesis has been undertaken by me. Technical and analytical help that I received from my colleagues and collaborators is listed in the acknowledgements section. Luise Marino July 2012 3 Abstract 4 Abstract Background: Specific nutrients such as glutamine have an important role in the management of critically ill patients, modulating the host response to stress, improving outcome. The mechanisms of action of glutamine are thought to be through the release of heat shock protein 70 (HSP70), which helps orchestrate an appropriate inflammatory response to critical illness. HSP70 release stimulates immune activation, maintenance of cell homeostasis, cellular protection, preventing apoptotic cell death and is associated with increased survival following severe trauma. However, much of the work considering the use of glutamine and HSP70 in critical illness has been completed in adults. The focus of the current research was therefore to investigate HSP70 release and its relationship to glutamine and inflammatory markers in healthy adults and paediatric volunteers using a whole blood endotoxin model. In addition to this, describing relationships between glutamine, HSP70 and inflammatory mediators, in a cohort of critically ill children. Methodology: An in vitro whole blood endotoxin stimulation model using lipopolysaccharide (LPS) was adapted for use in healthy adult (n=18) and paediatric volunteers (n=25) to investigate the effect of glutamine supplementation on the release of HSP70 and inflammatory mediators (IL-6, IL-8, IL-1β, IL-10, TNF-α). Plasma from children with acute meningococcal disease (MD) (n=143) and during convalescence (n=78) was analysed for levels of HSP70, inflammatory cytokines (IL-6, IL-8, IL-10, TNF-α) and glutamine. Following a clinical folder review, transcriptomic analysis of differential gene expression between acute disease and convalescence was carried out in a subset of children. Results: A whole blood stimulation model was effective in measuring the inflammatory response to endotoxin in vitro over an incubation period of 4 to 24 hours. Glutamine supplementation significantly increased HSP70 expression over time in healthy adult and paediatric volunteers compared to unsupplemented controls (p<0.05). HSP70 levels in vitro rose over time and was correlated with inflammatory mediators (IL-1β, TNF-α, IL-8) in adults, and IL-6, TNF-α in paediatric samples at 4 hours, following glutamine supplementation. There was no relationship between HSP70 and IL-10 in either adults or children. Glutamine supplementation significantly attenuated the release of IL-8 at 24 hours (p<0.05) and in the paediatric in vitro endotoxin model significantly increased TNF-α release at 4 hours 5 (p<0.005). Although there were no other significant differences in the release of inflammatory mediators (IL 6, TNF-α, IL10) between conditions, glutamine supplementation appeared to attenuate the release of inflammatory mediators in adults in contrast to promoting the release of inflammatory mediators in children. In patients with MD, HSP70 was significantly increased (p=<0.0001) in the acute phase [26.7ng/ml; SD± 79.95 (median 5.7ng/ml; range 0.09 – 600.5ng/ml)] vs. convalescence [mean 3.16ng/ml ±SD; 5.67 (median 1.045ng/ml range 0.001 – 37.31ng/ml)]. Mean glutamine levels during the acute phase were 0.31mmol/l; ±SD 0.13 (median 0.31mmol/l; range 0.50 – 0.64) and for convalescence 0.40mmol/l; ±SD 0.14 (median 0.40mmol/l; range 0.72 – 0.64). During the acute phase of illness, glutamine levels were 52% below the normal range, (using 0.6mmol/l as the lower reference range). In convalescent samples, taken on average 55 days later (35 – 135 days), glutamine levels were found to be 26% below normal range. Results from the transcriptomic analysis show that genes associated with HSP70 and inflammatory signalling pathways, glycolysis and gluconeogenesis are significantly upregulated and those relating to adaptive immunity are downregulated during the acute phase of meningococcal disease compared to convalescence. There were numerous correlations between plasma glutamine and HSP70 with genes associated with p38MAPK signalling pathway during the acute phase of meningococcal disease, suggesting that glutamine and HSP70 could mediate the inflammatory response to infection. Conclusion: Glutamine supplementation increased the release of HSP70 in an in vitro endotoxin model in children and adults. Children with MD during the acute phase of their illness have significant glutamine depletion, occurring early during the course of the disease, with a concomitant increase in plasma levels of HSP70 and inflammatory mediators. A transcriptomic analysis of differential gene expression showed that genes associated with HSP70 (HSPA1A and HSPA1B) were differentially upregulated during the acute phase of illness. HSP70 genes and plasma levels of HSP70 were correlated, which was suggestive of a role in meningococcal disease. Relationships were found with plasma levels of HSP70 and glutamine and genes associated with p38MAPK signalling pathway during the acute phase of meningococcal disease, suggesting that glutamine and HSP70 could mediate the inflammatory response to infection. Future work would aim to understand the effect of 6 glutamine supplementation on gene expression during critical illness, especially relative to the differential expression of genes associated with HSP70, inflammatory signalling pathways and those relating to the antigen presentation pathway. 7 Acknowledgements I would like to thank the Laventis Trust for providing the funding for this research project, which I hope will lead to the use of novel nutrition therapy as part of the medical management of critically ill children in the future. To my supervisor, Dr. Parviz Habibi for providing this amazing opportunity, along with supervisors Dr. Nazima Pathan and Dr. Rosan Meyer for their support, encouragement and guidance in the completion of this research project. Professor Michael Levin for generously providing access to samples from the meningococcal disease biobank. Dr. Victoria Wright for her assistance with laboratory techniques and related technical advice for the transcriptomic analysis of the meningococcal disease database. Professor Paul Langford for providing excellent academic mentorship and pragmatic advice. Dr Margaret Hancock and her team, Charing Cross Hospital, for their expertise and assistance in analysing the plasma glutamine samples. Yvonne Clements, Meso Scale Discovery with regards to training for MSD multiplex assays. Statistical Support Services at Imperial College for their assistance in the statistical analysis plan for the data arising from this project. To the healthy adult volunteers and St Mary’s Hospital Imperial NHS Healthcare Trust patients for partaking in this study. To my family and friends for their unwavering support and faith, but more especially to my wonderful daughter for her patience and understanding beyond her years. Finally, in memoriam to Professor Joe Ireland, Paediatric Gastroenterology, Red Cross War Memorial Children’s Hospital, Cape Town for being an inspirational mentor. 8 Table of Contents Abstract 5 Acknowledgements 8 Table of contents 9 List of Tables 18 List of Figures 20 Abbreviations 24 References 166 Appendices 194 Chapter 1: Introduction 26 1.1. Epidemiology and health economic burden of sepsis 27 1.2 Definition of SIRS, sepsis and septic shock 28 1.3 Causative pathogens 29 1.4 Pathophysiology of sepsis 30 1.5 Infectious process 31 1.6 Innate immune response during sepsis 32 1.7 Cytokine release 34 1.7.1 IL – 6 35 1.7.2 TNF-α 36 1.7.3 IL-8 36 1.7.4 IL-10 36 1.7.5 Cytokine release in N. meningitidis 37 1.7.6 Cytokine pathogenesis 38 1.8 HSP70 and the relationship to the immune and inflammatory response 38 1.8.1 Clinical consequences of upregulated HSP70 release 42 1.8.2 Upregulation and release of HSP70 into the extracellular milieu 43 1.8.3 The effect of the “model” on the inflammatory response 44 mediated by HSP70 1.9 Glutamine and the upregulation of the heat shock response in sepsis 47 1.9.1 Glutamine requirements in infancy 51 1.9.2 Glutamine and anabolism 51 9 1.9.3 Enteral vs. parenteral administration of glutamine 51 1.9.4 Use of glutamine in children 54 1.9.5 Mechanisms for glutamine supplementation on HSP70 expression 56 1.10 Novel therapeutic strategies in sepsis 57 1.11 Conclusion 59 1.12 Hypothesis 61 1.13 Research Aims 61 Chapter 2: Materials and Methods 63 2.1 Ethical consideration 64 2.1.1 In vitro experiments 64 2.1.2 Children with meningococcal disease 64 2.2 In vitro experiments 64 2.2.1 Optimisation phase 65 2.2.2 Experiment 1: To examine the effects of endotoxin and glutamine 66 using a whole blood model on HSP70 expression in healthy adult volunteers and its association with markers of inflammatory response in vitro 2.2.3 Experiment 2: To examine the effects of endotoxin, glutamine and 67 a HSP70 inhibitor KNK437, on HSP70 expression, using a whole blood model, in healthy adult volunteers and its association with markers of inflammatory
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