Characterisation of human macrophage functions in innate immunity Juliana Ariffin B. Biomedical Science (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 Institute for Molecular Bioscience Abstract Macrophages are key cellular mediators of the innate immune system. During an infection, phagocytosis of microorganisms delivers them to the macrophage phagolysosome where they are targeted for destruction by immediate antimicrobial responses. In parallel with this, signalling via pattern recognition receptors such as the Toll-like Receptors (TLRs) turns on expression of a set of genes to enable a second wave of inducible antimicrobial responses. Such responses enable macrophages to combat microorganisms that can evade immediate antimicrobial pathways. Mouse models have provided a powerful tool to study the innate immune system in the context of infection and inflammation, however some antimicrobial pathways are divergently regulated between human and mouse. This may partly reflect divergent evolution between species, due to selection pressure to co-evolve with rapidly-evolving pathogens. In view of this, this project aimed to explore novel aspects of human macrophage antimicrobial pathways. In Chapter 3 of this thesis, genetic analysis was conducted to validate the differential expression of novel TLR4-inducible genes in primary human versus mouse macrophages. From this initial analysis, four genes (RNF144B, BATF3, G0S2 and SLC41A2) were chosen for further functional analysis. In addition to their differential regulation, these genes were chosen based on novelty and biological functions in the context of innate immunity. Functional analysis by gene knockdown focused on investigating potential roles in macrophage inflammatory and antimicrobial responses. These studies led to the identification of the E3 ubiquitin ligase RNF144B as a novel regulator of NLRP3 inflammasome activation. Further analysis (Chapter 4) revealed that RNF144B broadly affected inflammasome function by mediating LPS inducible expression of the inflammasome substrate, IL-1β. Consequently, p21WAF1 was investigated as a potential RNF144B target that controls regulated IL-1β expression in human macrophages. The second approach to investigate novel human macrophage antimicrobial mechanisms was to examine the effect of pharmacological agents, currently under ii investigation as modulators of inflammation and innate immunity, on human macrophage antimicrobial responses against Gram-negative bacteria (Chapter 5). Previous studies utilising histone deacetylase inhibitors (HDACi) in mouse macrophages had demonstrated that HDACi impair macrophage phagocytosis and antimicrobial responses. However, I found that, while pre-treatment of primary human macrophages with HDACi did compromise phagocytic uptake of bacteria, co- treatment with HDACi actually enhanced clearance of S. Typhimurium and E. coli by promoting the generation of mitochondrial reactive oxygen species. The G protein- coupled receptor, C5aR, was also revealed to amplify antimicrobial responses in human macrophages via an ERK-dependent pathway. These pharmacological studies thus provided new insights into regulation of human macrophage antimicrobial responses against Gram-negative bacteria. In summary, findings from this thesis revealed RNF144B as a novel human-specific regulator of TLR-inducible IL-1β expression in macrophages, and the potential for HDACi and C5aR agonists to boost the clearance of intracellular pathogens by human macrophages. Thus, knowledge generated from this thesis provides new insight and avenues for future research on the molecular mechanisms that regulate both inflammatory and antimicrobial responses in human macrophages. iii Declaration by Author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated that contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during Candidature Peer reviewed papers: Review: 1) Ariffin, J.K., and Sweet, M.J. (2013). Differences in the repertoire, regulation and function of Toll-like Receptors and inflammasome-forming Nod-like Receptors between human and mouse. Curr Opin Microbiol, doi: 10.1016/j.mib.2013.03.002. Journal Article: 1) Vernon Seow, Junxian Lim, Abishek Iyer, Jacky Y Suen, Juliana K Ariffin, Daniel M Hohenhaus, Matthew J Sweet, David P Fairlie. (2013). Inflammatory Responses Induced by Lipopolysaccharide Are Amplified in Primary Human Monocytes but Suppressed in Macrophages by Complement Protein C5a. Journal of Immunology. 15;191(8):4308-16. doi: 10.4049/jimmunol.1301355. Book chapter: 1) Ronan Kapetanovic, Juliana K. Ariffin, Matthew J. Sweet (2014). Evolutionary Divergence in Human Versus Mouse Innate Immune Gene Regulation and Function. Pontarotti, Pierre (Ed.) EvolutionaryBiology: Genome Evolution, Speciation, Coevolution and Origin of Life. doi: 10.1007/978-3-319-07623-2_6. v Conference abstracts Oral conference presentations: 1) Characterization of human-restricted TLR4 responses identifies a novel ubiquitin ligase required for NLRP3 inflammasome responses in human macrophages. Juliana K. Ariffin, Kate Schroder, Matthew J. Sweet. Presented at the 44th Annual Scientific Meeting of the Australiasian Society for Immunology (ASI), 1-4 December 2014, Wollongong, NSW, Australia. Poster presentations: 1) Characterization of human-restricted TLR4 responses identifies a novel ubiquitin ligase required for NLRP3 inflammasome responses in human macrophages. Juliana K. Ariffin, Kate Schroder, Matthew J. Sweet. Cytokines Down Under Conference, 26-29 October 2014, Lorne, Melbourne, Australia. 2) Characterization of human-restricted TLR4 responses identifies a novel ubiquitin ligase required for NLRP3 inflammasome responses in human macrophages. Juliana K. Ariffin, Kate Schroder, Matthew J. Sweet. Lorne Infection and Immunity Conference, 19-21 February 2014, Lorne, Melbourne, Australia 3) Novel Human Macrophage Antimicrobial Mechanisms. Juliana K. Ariffin, Nilesh J. Bokil, Vernon Seow, Kate Stacey, David Fairlie, Kate Schroder, Matthew J. Sweet. Brisbane Immunology Group, 16-17 August 2012, Kingscliff, New South Wales, Australia. 4) Novel Human Macrophage Antimicrobial Mechanisms. Juliana K. Ariffin, Nilesh J. Bokil, Vernon Seow, Kate Stacey, David Fairlie, Kate Schroder, Matthew J. Sweet. UQ-SBMS Postgraduate Symposium, The University of Queensland, 24-26 August 2012, Brisbane, Australia. vi Publications included in this thesis Ariffin, J.K., and Sweet, M.J. (2013). Differences in the repertoire, regulation and function of Toll-like Receptors and inflammasome-forming Nod-like Receptors between human and mouse. Curr Opin Microbiol, doi: 10.1016/j.mib.2013.03.002. Partially incorporated as paragraphs in Chapter 1. Table 1, Figure 1 and Figure 2 from this review were also included as Table 1.2, Figure 1.1 and Figure 1.2 in this thesis. Contributor Statement of contribution Ariffin, J.K. Wrote and edited manuscript (50%) Sweet, M.J. Wrote, edited and reviewed manuscript (50%) Vernon Seow, Junxian Lim, Abishek Iyer, Jacky Y Suen, Juliana K Ariffin, Daniel M Hohenhaus, Matthew J Sweet, David P Fairlie. (2013). Inflammatory Responses Induced by Lipopolysaccharide Are Amplified in Primary Human Monocytes but Suppressed in Macrophages by Complement Protein C5a. Journal of Immunology. 15;191(8):4308-16. doi: 10.4049/jimmunol.1301355. Data used to generate Figure 8 in this paper was included in this thesis as Figure 5.11 and Figure 5.12. Contributor Statement of contribution Vernon Seow Data acquisition and analysis, writing and editing of manuscript (45%) Junxian Lim Provided experimental reagents, reviewing of manuscript and analysis (5%) Abishek Iyer Project direction, reviewing of manuscript (5%) Jacky Y Suen Project direction, reviewing of manuscript (5%) Juliana K Ariffin Data acquisition and analysis (5%) Daniel M Hohenhaus Provided experimental reagents (2%) Matthew J Sweet Edited and reviewed manuscript (10%) David P Fairlie Project conception, edited and reviewed manuscript (23%) vii Contributions by others to the thesis Assoc. Prof. Matthew Sweet made significant contributions to the conception and design of these projects as well as interpretation