Characterisation and therapeutic modulation of toll-like receptor signalling in response to the intracellular pathogen F. tularensis
The induction of an innate immune response upon infection is dependent on the detection of the invading organism and the generation of a signalling cascade leading to the production of inflammatory mediators. Toll-like receptors are expressed on multiple cell types and induce the activation of a complex network of signalling pathways containing numerous branches with multiple interactions and cross-talk between the different branches. The TLR system is integral to the generation of a protective immune response and as such is an important target for pathogen-associated modulation. Many bacterial and viral pathogens employ strategies for interrupting or modulating TLR signalling to evade the host immune response. The obligate intracellular bacterial pathogen, F. tularensis, successfully invades and replicates within immune and epithelial cells. However, despite significant research the exact mechanisms used by this pathogen to successfully evade the host immune response remain elusive. To establish the exact signalling events that occur within a host upon infection with F. tularensis, the activation of specific signalling proteins was characterised using in vitro and in vivo models. The MAPKs, ERK and p38, were identified as critical in generating the host response. Furthermore, the temporal regulation of these signalling proteins was found to be bi-phasic with an early transient activation of both ERK and p38 followed by a sustained activation of ERK and a suppression of p38 activation at later time points. The role of ERK was investigated further using a specific inhibitor (PD0325901). Although there was no decrease in bacterial burdens in vitro and no increase in survival in mice treated with PD0325901, the inhibition of ERK activation reduced the secretion of TNF and IL-6 and reduced systemic bacterial proliferation in vivo. The induction of immune signalling cascades requires the activation of one or more receptors. The contribution of TLR2, TLR4 and TLR9 to the immune response to F. tularensis infection was examined using KO cell lines and specific antagonists. TLR2 was confirmed as a receptor for F. tularensis and was observed to play a role in the translational regulation of TNF. A role for TLR4 was also identified and further characterisation identified a potential priming relationship with TLR9. Sub-stimulation of 13 TLR4 by LPS enhanced the response induced by a subsequent stimulation of TLR9 by purified F. tularensis DNA. Overall, this study has provided evidence that, during infection, F. tularensis interacts with innate immune signalling pathways. By simultaneously suppressing p38 activation and prolonging ERK activation F. tularensis is able to regulate cytokine secretion and the induction of host-cell death mechanisms. Furthermore, this work has demonstrated that the activation of TLR9 by F. tularensis genomic DNA can be primed by a prior sub-stimulation of TLR4, although more research is required to fully understand the contribution of this interaction to the pathogenesis of F. tularensis.
Francisella, Toll-like receptor, Tularensis
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