Characterisation of human macrophage functions in innate immunity

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 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 primaryhuman macrophages with HDACi did compromise phagocytic uptake of bacteria, cotreatment with HDACi actually enhanced clearance of S. Typhimurium and E. coli by promoting the generation of mitochondrial reactive oxygen species. The G proteincoupled 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.