Determinants of Tuberculosis Severity

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 DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a leading cause of death from infection worldwide. The interaction between Mycobacterium tuberculosis (Mtb) and host macrophages (MΦ) lies at the heart of TB pathogenesis. Our preliminary data indicate that virulent Mtb suppresses a host-protective apoptotic response of infected MΦ in order to use these cells as a protected replication niche. After growing to an optimal intracellular bacillary load, Mtb then induces MΦ necrosis that allows bacteria to escape and spread to naïve phagocytes. We propose that infection-induced MΦ necrosis is the predominant source of signals that recruit neutrophils to TB lesions. Neutrophils likely enhance host defense when TB disease is constrained but when Mtb replication is poorly controlled or when conditions exist that reduce the threshold for MΦ necrosis, neutrophils may accumulate in excess of clearance capacity and die in situ. This establishes a feed-forward mechanism for progressive inflammation and lung tissue damage mediated by neutrophil-derived hydrolases. On this basis we plan to identify the bacterial gene(s) required by Mtb to trigger MΦ necrosis and to identify the specific signals linked to MΦ necrosis that lead to the accumulation of neutrophils at sites o TB disease in the lung. Our preliminary data suggest that one or more genes in the Mtb PhoPR regulon are required for this high bacterial load "burst size" MΦ cytolysis. In Aim 1 we will leverage that finding by systematically testing deletion mutants of candidate PhoPR-regulated genes for loss of cytolytic function. These mutants will be generated using state of the art recombineering methods in collaboration with Dr. Christopher Sassetti (UMass). As a complementary approach we will perform an unbiased screen of an Mtb transposon library for the loss of cytolytic function phenotype. Aim 2 investigates the signaling mechanisms linking MΦ necrosis to neutrophil recruitment, here leveraging new knowledge about the role of leukotriene B4 and 12/15-lipoxygenase in recruiting neutrophils to local sites of cell damage. Aim 3 investigates the relationship between neutrophilic inflammation and tissue injury in the mouse aerosol TB model and in samples of bronchoalveolar lavage fluid and cells obtained from pulmonary TB patients made available through collaboration with Dr. Xinchun Chen (Shenzhen Third People's Hospital and Shenzhen-Hong Kong Institute for Infectious Diseases). Based on the linked hypotheses that Mtb-induced MΦ necrosis promotes neutrophil trafficking to the lung and that neutrophils are dominant mediators of tissue injury, this project has the potential to reveal new targets for pathogen and host-directed therapies to limit neutrophilic inflammation and pulmonary impairment in TB.