Effects of sodium benzoate on the innate immune response to gram-negative bacteria and Toll-like receptor stimulation By Andre Luis Ribeiro Ribeiro A thesis submitted to UCL for the degree of Doctor of Philosophy Eastman Dental Institute Department of Microbial Disease 2018 I, Andre Luis Ribeiro Ribeiro confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 non scholae sed vitae discimus 3 Abstract Sodium benzoate (NaB) is a sodium salt that is widely used in the pharmaceutical, cosmetic and food industries. This widespread use results in almost everybody in the world being exposed daily to this compound. Currently very little is known about the effects of NaB can have on the immune response, even though it has been associated with the clinical course of chronic inflammatory diseases, such as orofacial granulomatosis and neurodegenerative disorders. Here, I show that THP-1 cells, a monocytic human cell line presents an altered immune response when exposed to NaB. Immunologically stimulated THP-1 cells in the presence of NaB secreted reduced levels of IL6 and IL1β and higher levels of TNF, while other cytokines such as IP10 and IL8 were unaffected. The inhibitory effect in IL6 and IL1β secretion was a consequence of a free radical scavenging characteristic of NaB, which neutralizes the reactive oxygen species (ROS) generated downstream of TLR activation. This resulted in the impairment of a secondary signalling event, which is required to fully activate the cells immune response. The use of microarray analysis in combination with q quantitative proteomic analysis revealed that NaB has a significant effect on the THP-1 cells beyond the alteration in cytokine secretion. NaB also interferes with cellular amino acid metabolism and has a major attenuating effect on the immune response. Taken together, these results suggest that NaB is not inert and has a major effect on a cells ability to mount an immune response. These findings could have major implications in how NaB is used in the future and in particular if it can be beneficial as a treatment for chronic inflammatory diseases, such as diabetes, atherosclerosis, rheumatoid arthritis, neurodegenerative disorders, and so on. On the other hand, by disturbing the inflammatory response, NaB could have a negative impact on other conditions such as orofacial granulomatosis. Further work will be needed to determine the role NaB plays in human inflammatory diseases. 4 Impact statement Sodium benzoate (NaB) is widely used by pharmaceutical, cosmetic and food industries, resulting in a routinely daily exposure to most people worldwide. It is added to processed food as a preservative to control microbial growth and can be found in soft drinks, fruit juices, beers, salad dressings, but also occurs naturally in many fruits and vegetables: apples, tomatoes, strawberries, and dark green vegetables. An individual’s exposure to NaB has the potential to be very high and cumulative due to the diverse sources of ingestion. Even though our ingestion of this compound is so high we know very little about how NaB affects the immune response. NaB has been associated with chronic inflammatory diseases, such as orofacial granulomatosis (OFG) and neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease. My findings will have the potential to have an impact on a whole spectrum of industries that routinely include NaB in their products. The Oral Medicine department in the Eastman Dental Hospital is a reference service in the country to treat patients with OFG, and even though using all the resources available, only 70% of the patients had a complete resolution of their problems in a 10-year period. The treatment is usually based on immunomodulatory drugs, which have many side effects. Studies have shown that changing the patient diet for a benzoate-free type without any medication, achieves a resolution in between 50-70% of the cases, which is not too different to that observed with current medications. My results provide a plausible mode of action for NaB and how it may influence disease state in OFG. Therefore, the implementation of benzoate free diets may be used more frequently for this group of patients. My results may have an impact on the regulatory bodies and governments that conduct routine toxicity tests on food additives and set national and international guidelines. The toxicity tests prior to approval to human consumption are usually made in a controlled environment under physiological conditions. However, in everyday life, people are exposed to many stimulatory substances and microbial agents that can stimulate the immune system and alter metabolic processes. Although the regulatory agencies require the companies to describe what is in their products 5 (benzoates in Europe are under the numbers E210-E219), the population should be aware of the negative effects associated with these substances. My work has also demonstrated that using techniques such as microarray and proteomics has the potential to improve our understanding of how a compound can influence the cellular response in a non-biased way. NaB (or any other substance capable of neutralizing ROS) which reduce the release of IL6 and IL1β can potentially work as a therapy for many chronic inflammatory diseases such as diabetes, atherosclerosis, rheumatoid arthritis, neurodegenerative disorders, and so on. It is therefore possible that my results will have an impact on a diverse array of clinical specialities and researchers who specialize in the treatment of these conditions. 6 Contents Table of Figures ..................................................................................................................... 11 Table of Tables ....................................................................................................................... 14 Acknowledgements .............................................................................................................. 15 Statement of Collaborative Work .................................................................................... 17 List of Abbreviations ............................................................................................................ 18 Chapter 1 Introduction ...................................................................................................... 23 1.1 Sodium benzoate (NaB) and benzoic acid ............................................................. 23 1.1.1 Antimicrobial mechanisms of benzoates ...................................................... 26 1.1.2 Food spoilage ........................................................................................................... 30 1.2 Sodium benzoate and human diseases ................................................................... 31 1.2.1 OFG and sodium benzoate .................................................................................. 31 1.2.2 Neurodegenerative disease and sodium benzoate ................................... 41 1.2.3 Urea cycle disorders (UCD) and sodium benzoate ................................... 46 1.3 The immune system (General overview) ............................................................... 50 1.3.1 Anti-bacterial recognition and early response ........................................... 51 1.3.2 Toll-like receptors .................................................................................................. 51 1.3.3 Reactive oxygen species (ROS) ......................................................................... 61 1.4 Summary of investigations conducted and hypotheses ................................... 64 Chapter 2 Materials and methods .................................................................................. 65 2.1 Cell culture .......................................................................................................................... 65 2.1.1 THP-1 cells ................................................................................................................ 65 2.1.2 Peripheral blood monocyte-derived macrophages (PBM-DM) ........... 65 2.2 Reagents for biological assays .................................................................................... 66 2.2.1 NaB ............................................................................................................................... 66 2.2.2 Heat-killed bacteria ............................................................................................... 67 2.2.3 Mixed biological reagents ................................................................................... 68 2.3 Cell stimulation ................................................................................................................. 69 2.3.1 NaB treatment.......................................................................................................... 69 2.3.2 Stimulation of cells ................................................................................................. 69 2.4 Cell viability assays ......................................................................................................... 70 2.4.1 MTT cell viability assay ........................................................................................ 70 2.4.2 CCK-8 cell viability assay ..................................................................................... 71 2.5 pH measurements ...........................................................................................................