Effects of heat labile Enterotoxin B subunit on murine dendritic cells
Creators: name: Ji, Jing
Publisher: name: The Australian National University
Year: 2014
Subjects: subject: Enterotoxins Research
subject: Dendritic cells
subject: Escherichia coli

description: The potent immunomodulatory properties of the B subunit of E. coli heat labile Enterotoxin (EtxB) have not been fully investigated. While the effect of EtxB on monocytes, macrophages, B cells and T cells has been addressed, no work has been undertaken to date on how dendritic cells (DCs) respond to EtxB. Dendritic cells are the most potent professional antigen presenting cells, which bridge the divide between innate and adaptive immune responses. To further explore mechanisms of EtxB immunomodulation, this thesis investigates the effect of EtxB on murine DCs. EtxB was purified using multiple ion exchange columns, an endotoxin removal column, and an alkanediol wash, to achieve endotoxin minimisation. The final endotoxin level was 0.04EU/ug, below that of previously published work. Immature and mature murine DCs were characterised based on marker expression information in the literature as CD8+ conventional DCs (cDCs), CD8- cDCs, plasmacytoid DCs (pDCs), cDC precursors and pDC precursors, with subset distribution assessed following in vivo and in vitro EtxB treatment. Cell viability was measured using propidium iodide (PI) staining, with MHC-II, CD80, CD86 and CD69 marker expression measured to gauge DC activation. Changes in the expression level of 84 genes in CD8- cDCs were assessed, resulting in 7 genes downregulated (Ccl11, Ccr1, Cd1d1, Cd2, Icam2, Cxcr1, Inhba) and 1 gene upregulated (Lyn) following EtxB treatment. Murine chimeras were reconstituted with bone marrow cells from CD11c-DTR/GFP transgenic mice in which the CD11c promoter controls a fusion protein of diphtheria toxin receptor/green fluorescent protein. These chimeras were utilised to investigate the requirement for DCs in EtxB-mediated T cell activation. The action of EtxB as an adjuvant has been investigated using molecular and cellular techniques. In particular, this is the first study to demonstrate that EtxB requires DCs for adjuvant function, and validates further investigation on the specific effect of EtxB on DC subset development and function in immunity. EtxB was shown to activate immature Flt3L-derived DCs, by increasing expression of the costimulatory molecules CD80, CD86 and MHC-II, and by decreasing cell viability. In contrast, CD80, CD86, CD69 and MHC-II marker expression levels were reduced on mature DCs following EtxB treatment, possibly as a result of 'overactivation'. This was also associated with an increase in DC viability, a property which could enable prolonged antigen presentation. This Thesis has taken snapshots of DC responses at various time points both in vivo and in vitro following EtxB treatment. DCs have been shown to react in different ways to EtxB depending on the stimulants used to drive DC production, the activation state of DCs as immature versus mature cells, cell turnover, and duration of antigen exposure. The impact of EtxB on immune responses appears to be mediated largely through DCs, although the mechanisms seem complex and subject to the dynamic role of DCs. In summary, the adjuvant effect of EtxB appears to be manifest by EtxB having a modulating effect on DC activation and turnover, allowing prolonged antigen presentation to T cells.