Do post-receptor binding events decide the fate of mycobacteria in bovine macrophages?

Bovine tuberculosis (TB) is a disease of cattle caused by the bacterium Mycobacterium bovis (M.bovis), which has severe consequences for animal welfare in addition to financial implications. In the UK, the number of cases of bovine TB continues to increase, despite the application of control measures. Vaccination may be a better means of control, compared to the skin test that is currently used to identify infected cattle which have to be destroyed to prevent spread of disease. The vaccine BCG (derived from M.bovis) is currently used against TB in humans, and has been tested for its suitability in preventing bovine TB. As in humans, BCG use in cattle has been shown to be partially protective against disease. At present, BCG is the best vaccine against TB, but we do not completely understand why infection with M.bovis results in disease and infection with BCG results in protection. Interestingly, M.tuberculosis, which causes TB in humans, does not cause disease in cattle, while M.bovis is able to cause TB in cattle and humans. Antigen presenting cells (APC) are the first cells of the body's internal defence system (the immune system) to come into contact with disease-causing bacteria (pathogens). APC are responsible for the onset of the response of the immune system towards controlling and eliminating pathogens. Pathogens are recognised by APC through special molecules on their surface, known as pattern recognition receptors, which play a vital role in the development of the immune response. However, mycobacteria are able to influence the response of APC, so rather than killing the pathogen the APC's defence mechanisms are altered - allowing the bacterium to survive, multiply and go on to cause disease. The interaction between the APC and mycobacteria triggers a series of events, known as signalling cascades, resulting in the expression of molecules that determine whether APC will initiate a protective response or allow the progression towards disease. This proposal aims to study one of the major APC, called the alveolar macrophage. Residing in the lung, this cell is one of the first APC to come into contact with bacteria invading the body through the respiratory route. We will study how this bovine cell interacts with the TB-causing cattle strain M.bovis (which also causes TB in humans), BCG (vaccine strain) and M.tuberculosis (which causes disease in humans but not cattle). By comparing the response of the cell to the three different bacteria, we aim to identify alveolar macrophage responses only seen with the TB causing cattle strain, M.bovis. This should allow us to identify stages in the immune response where a particular reaction is critical if the bacterium is then going to cause disease rather than immunity, as seen with BCG and M.tuberculosis in cattle. This proposal aims to test the hypothesis that M.bovis ensures its survival within bovine alveolar macrophages and, as a consequence the progression of disease, by specific pattern recognition receptor usage and interference with the signalling cascades - influencing the outcome of the immune response. This contrasts with the receptors used and signalling cascades activated by BCG or M.tuberculosis, leading to the secretion of specific molecules by the infected cells that result in immunity rather than active disease. We propose that the difference in host specificity of M.bovis (includes cattle and humans) and M.tuberculosis (host specificity - only humans) and the virulence (ability to cause disease) of M.bovis (causes TB) and BCG (no disease, can develop immunity) in the bovine alveolar macrophage are key to the response of the cell and subsequent bacterial killing, and consequently critical to the outcome of exposure to the mycobacteria. A better understanding of the mechanisms in bovine alveolar macrophages that lead either to protective immunity or allow disease to develop is important for the development of novel therapies and vaccines.

Despite control measures, the prevalence of bovine tuberculosis (TB), caused by Mycobacterium bovis, is increasing, enhancing the risk of zoonotic transmission. M.bovis (Mb), closely related to M.tuberculosis (MTB), also causes disease in humans. These mycobacteria are virulent in their primary target species, but have different host ranges. MTB is severely host-adapted, causing disease in humans/primates only. Little is known about the mechanisms behind this host specificity and resistance of cattle to MTB. Vaccination is the most effective and sustainable way of preventing the spread of disease. BCG, attenuated Mb, confers a degree of protection in cattle. This study exploits the ability to investigate differences in virulence and pathogenicity in an animal species that is the natural host in one case and shows innate resistance in another, identifying mechanisms that could be targeted as features of effective vaccines. The first antigen presenting cells (APC) that come into contact with inhaled mycobacteria are the alveolar macrophages (AlvM) in the lung. Conserved pattern recognition receptors (PRR) on these cells influence recognition, ingestion, activation of signalling pathways and processing. Virulent mycobacteria influence the cellular response of AlvM, facilitating intracellular survival. This can occur on two levels - usage of different PRR and stimulation of alternative signalling cascades. Preliminary data showed differences in the response of bovine AlvM (boAlvM) to these three bacteria. We propose that the differences in host specificity of Mb and MTB and the relative virulence of Mb and BCG in the boAlvM are key to the cell's response, subsequent bacterial killing, and therefore critical to the outcome of exposure. This proposal tests the hypothesis that virulent Mb facilitates its survival within boAlvM (and the progression of disease) by Mb-specific PRR usage and subsequent interference with the signalling cascades - influencing the outcome of the ensuing adaptive immune response. This contrasts with the PRR used and signalling cascades activated by BCG/MTB, for which secretion of specific immune modulators by infected cells results in immunity. To investigate whether Mb/BCG interacts with PRR on boAlvM in a host adapted manner that differs from MTB, we will block mycobacterial receptors prior to bacterial exposure. TNF (ELISA) and NO production (Griess assay) will be assessed and intracellular bacterial survival determined. Having identified the PRR used, we will determine the AlvM signalling pathways activated by mining microarray data from the VTRI project, comparing it to data from Mb and MTB-infected AlvM. By analysing whether differing pathogenicities and virulence of Mb, BCG and MTB can be attributed to blocking steps in the MAPK pathway, we will test the hypothesis that more virulent strains limit MAPK activation, resulting in decreased production of inflammatory mediators. We will analyse the kinetics of protein phosphorylation (by western blotting), and the effects of specific MAPK inhibitors. Having shown that TNF, NO and CXCL8 and are produced to varying extents by boAlvM exposed to Mb, BCG and MTB, the effects of protein kinase inhibitors on their production following boAlvM infection will be analysed. Using inhibitory RNA (RNAi) to block key molecules in signalling pathways, we can confirm the role of these pathways in AlvM activation by Mb, BCG or MTB, subsequent bacterial survival and host response, and potentially alter the cells' response to, and outcome of, infection. The effects of RNAi will be monitored by western blotting and real-time PCR. Overall, the described experiments will provide an understanding of the boAlvM activation program by different mycobacterial strains and the modulation of the signalling machinery induced by them. This information will be vital in designing vaccines and cytokine therapies that engage the innate immune system in a targeted fashion.