Modelling bovine tuberculosis infection in stem cell-derived macrophages

Abstract

Bovine tuberculosis is a serious disease in cattle which has significant economic repercussions and poses a risk to human health. The pathogen responsible, Mycobacterium bovis, is inhaled into the lungs and engulfed by alveolar macrophages (AMs). The early interactions between M. bovis and the AMs are complex and are crucial in determining the outcome of infection. Whilst the AMs have numerous mechanisms to eliminate pathogens including the production of antimicrobials and cytokines, M. bovis can subvert these processes and create an environment within the AM where the bacteria can thrive. It is important to investigate M. bovis- macrophage interactions as this may lead to the identification of potential targets for intervention. In order to study these interactions, a suitable cell culture model is required which resembles the macrophages found within the calf lung as closely as possible. Here, an efficient and reliable system has been developed to study M. bovis infection using bovine embryonic stem cell-derived macrophages (ESCdMs).

Ex vivo AMs obtained from calf bronchoalveolar lavage (BAL) fluid were analysed extensively so that the in vitro ESCdMs could be compared to them. Flow cytometric analysis demonstrated that BAL cells differentially expressed the scavenger receptor, CD163, and high CD163 expression corresponded with increased expression levels of other macrophage molecules compared to the CD163- subset. The BAL cells were shown to have uniformly high expression levels of CD172a, ADGRE1, CD206, and CD14, whilst there were lower levels of CD80, MHC II, CD1b, CD40 and CX3CR1. Analysis of BAL cells by single cell RNA sequencing (scRNASeq) showed that subsets of AMs exist which express genes involved in the cell cycle, antigen presentation, the inflammatory response, and host defence. The analysis of bovine BAL cells by both flow cytometry and scRNASeq indicated that subsets of macrophages exist within the lung which may have differences in ontogeny, microbial exposure history, lung localisation, or function.

Bovine ESCs provide unlimited, experimentally tractable macrophages when put through a three-phase differentiation protocol which involves mesoderm induction, myeloid lineage commitment and macrophage maturation. RT-qPCR analysis confirmed that pluripotency gene expression decreased and macrophage gene expression increased during the differentiation process, and RNASeq analysis demonstrated that ESCdMs and BAL cells expressed many of the same macrophage genes. Flow cytometric analysis indicated that bovine ESCdMs had high cell-surface levels of molecules involved in pathogen recognition and antigen presentation including CD206, CD163, CD14, MHC II, CD80, CD86, and CD1b. However, there was very low expression of CD172a and ADGRE1, which were highly expressed on the bovine BAL cells. The ESCdMs had functional similarities to BAL cells as both cell types were capable of phagocytosing fluorescent particles and both increased cytokine gene expression when exposed to lipopolysaccharide (LPS).

The IL10 gene encodes an important immunosuppressive cytokine which can be upregulated by M. bovis to enhance infection. Therefore, bovine ESC clones carrying a deletion in the IL10 gene were generated by CRISPR/Cas9 gene editing. These mutant ESCs were then differentiated into macrophages which were deficient in IL-10 to examine the impact of this cytokine in M. bovis- infected macrophages. Flow cytometric analysis of M. bovis-infected BAL cells, wild type ESCdMs, and IL10 knockout ESCdMs demonstrated that CD80, CD206, and CD40 expression increased during infection. However, differences between BAL cells and ESCdMs were also observed, and possible roles for IL-10 were identified. The presence of mycobacteria within the ESCdMs was visualised using rhodamine B-stained M. bovis. This showed that by 1 hour post infection most ESCdMs were free of M. bovis, but a small proportion of ESCdMs were more heavily infected and appeared to be in the process of dying.

The work presented in this thesis demonstrated that bovine ESCdMs can be generated and they are comparable to ex vivo macrophages. The use of the ESCdMs as a tool to model M. bovis infection should provide further insights into macrophage-mycobacteria interactions and identify targets for intervention.