The effects of Pseudomonas aeruginosa quorum sensing signalling molecules on human T cell function

Quorum sensing signalling molecules (QSSMs) are important to Pseudomonas aeruginosa virulence and biofilm development which aid establishment and persistence of these bacteria in the host. Recent progress in quorum sensing (QS) research has demonstrated that the two QSSMs, 3-oxo-C12-HSL and PQS interact with eukaryotic cells and modulate immune responses. Early research has indicated these two QSSMs are immunosuppressive, and because T cells play an important role in defending the host against the attack of P. aeruginosa (Stevenson et al., 1995), this warrants investigations into the interactions between QSSMs and T cells. Previous studies have shown 3-oxo-C12-HSL and PQS can exert differential immune-modulatory effects on mammalian immune responses, however, no studies have confirmed these activities using pure human T cells. The purpose of my PhD was to investigate for the first time, the effects of these two QSSMs on pure human T cells in a staged manner, beginning with mouse splenocytes, human peripheral blood mononuclear cells (hPBMCs) and finally pure T cells, then if successful, paving the way for gene array technology. This present work confirms inhibitory effects by QSSMs on mouse splenocytes stimulated to proliferate using the lectin concanavalin (ConA) or anti-CD3 antibody, and hPBMCs stimulated to proliferate using anti-CD3 and anti-CD28 antibody. In order to further understand interactions between QSSMs and the immune system, the effects of 3-oxo-C12-HSL and PQS on pure human T cell proliferation and cytokine production following stimulation of T cells with monoclonal antibodies directed against CD3 and CD28 were compared, using CsA as the positive control. All three compounds inhibited pure T cell proliferation. CsA and PQS were the more potent anti-proliferative compounds with IC50 values of 3.2±0.31 µM and 3.8±0.15 µM respectively compared to 19±1.62 µM for 3-oxo-C12-HSL, indicating the QSSMs ability to suppress T cell activity and therefore advantageous to P. aeruginosa. To further comprehend the mechanism of action of these two QSSMs, the effects of QSSMs on cytokine production were assessed. 3-oxo-C12-HSL significantly inhibited IL-2 release while PQS enhanced the production of IL-2 even though suppression of T cell proliferation was observed, suggesting a cytostatic effect and demonstrating PQS may in fact act proximally to the IL-2 receptor (IL-2R) or downstream of the T cell signalling pathway, whereas 3-oxo-C12-HSL acts on early T cell signalling events. 3-oxo-C12-HSL also inhibited production of IFNó in pure T cells and although results were inconclusive for PQS in pure T cell assays, both QSSMs were shown to have an inhibitory effect on IFNó in mouse splenocytes, suggesting suppression of T cell proliferation is via Th1. Furthermore, 3 oxo C12 HSL suppressed IL-4, IL-5, IL-10 and TNFñ while PQS suppressed IL 10 release at 3.12 µM and enhanced TNFñ release, indicating these QSSMs may inhibit T cell proliferation by eliminating both Th1 and Th2 response. The immune suppressive properties of 3-oxo-C12-HSL and PQS show potential as future therapeutic entities. Immunosuppressive drugs such as CsA and rapamycin are routinely used to maintain transplants and treat auto-immune disorders. However, they can be non-selective and are limited by their side effects including nephrotoxicity and neurotoxicity. Despite recent developments of new immunosuppressants, there remains an unmet need for less toxic and more widely applicable immunosuppressive agents. 3-oxo-C12-HSL and PQS are worthy of attention as possible future immunosuppressive agents used in conjunction with or in place of present immunosuppressants. In summary, this study clearly demonstrates for the first time that the two structurally diverse QSSMs, 3-oxo-C12-HSL and PQS, can exert differential modulatory effects on pure T cells, opening a path for further study into their mode of actions within the T cell signalling pathways and their effects at an RNA level.EThOS - Electronic Theses Online ServiceGBUnited Kingdo