Tuberculosis is a clinically significant disease, caused by Mycobacterium tuberculosis. Mycobacterial species subvert the host immune system to reside in macrophages, which are a major cellular mediator of innate immunity. Trehalose-6,6’-dimycolate (TDM) is a component of the mycobacterial cell wall and is a critical mediator of tuberculosis pathology in animal models. TDM plays an important role in host colonisation, and consequently in the pathology of disease, by mediating mycobacterial survival and the induction of inflammatory responses by macrophages. Despite its importance, there is a lack of clear perspective on the molecular mechanisms that mediate these TDM-induced macrophage responses, and how these might influence interaction of the host and pathogen. Furthermore, the majority of TDM in vitro studies have been performed with mouse macrophages, and as such little is known of the human innate immune response to TDM. This thesis addressed the role of a newly identified TDM receptor, Macrophage inducible C-type Lectin (Mincle), in the early host response to TDM, and in particular its function in orchestrating inflammatory responses, utilising a mouse model. Furthermore, the work presented here investigated the effects of TDM-stimulation on primary human cells.
Findings presented in this thesis show that Mincle mediated the expression of a subset of genes associated with mouse macrophage activation, and inferred a role for Mincle in the induction of inflammatory feedback loops. In depth investigation of TDM-induced changes in gene expression in the presence or absence of Mincle indicated a predominant role for this receptor at late (>12hr) time-points. This work further described a role for Mincle in the transcriptional regulation of G-CSF and CCL5 in mouse macrophages. Mincle was also demonstrated to be required for TNF release in response to TDM, via an alternative mechanism that increased the probability of Tnf mRNA translation, but did not significantly affect Tnf mRNA expression. Further investigation demonstrated that the role of Mincle in TNF secretion was independent of MAPK signalling, despite MAPK activation contributing to TNF secretion. In contrast to stimulation with TDM, Mincle was not essential for mediating the release of inflammatory molecules in response to infection with whole mycobacteria, nor was Mincle important for limiting mycobacterial growth in cultured mouse macrophages.
In contrast to the slow transcriptional program induced in mouse macrophages, RNA-Sequencing experiments revealed a rapid and broad transcriptional activity in human monocytes responding to mycobacterial pathogen associated molecular patterns (PAMPs). This study provides the first comprehensive description of early human gene expression events triggered in response to TDM, and furthermore made comparisons with the response to whole mycobacteria. The data indicated that TDM stimulation mirrored the majority of gene expression events induced by whole mycobacteria, implicating TDM as a key participant in the early human monocyte response to mycobacteria. Furthermore, TDM influenced the expression of many genes important to pathogen recognition, host cell survival and formation and maintenance of granulomas. This data demonstrates that TDM recapitulates the majority of the host response to mycobacteria.
The data in this thesis provides strong evidence that Mincle-TDM interactions represent only a single component in a broader host immune engagement strategy employed by the pathogenic mycobacteria. In hindsight, when considering the high level of redundancy repeatedly shown across many different knockout mouse models in the context of mycobacterial challenge, it is perhaps not surprising that Mincle also plays a redundant role in mediating mycobacterial responses. However this thesis also raises the spectre that current mouse models may not fully reflect the molecular processes at play in human innate immune cells. Certainly the human expression study conducted as part of this thesis suggests a significant contribution of TDM in the early monocyte response to mycobacteria, and it could be inferred that Mincle may have an important role during early human responses to mycobacterial infection. It is envisaged that future work regarding the role of Mincle in mycobacterial infections or host responses to TDM should focus on human innate immune cells.