David M. Tobin - US grants

DESCRIPTION (Provided by the applicant) Abstract: Tuberculosis (TB) kills nearly two million people annually. Long treatment regimens result in patient non-compliance, and the consequent development of drug-resistance. The HIV/AIDS pandemic has further hastened the emergence of extensively drug-resistant (XDR) strains that are both lethal and transmissible. New therapeutic approaches are urgently needed. Existing anti-TB therapies target the bacteria. This project will exploit my recent discoveries about host susceptibility to TB to develop novel therapeutic strategies based on targeting specific, genetically-determined host immune pathways. Through a large-scale forward genetic screen in the zebrafish I identified the leukotriene A4 hydrolase (lta4h) gene as critical in controlling susceptibility to TB. In fish and in mammals, the LTA4H enzyme functions as a rheostat, controlling relative levels of specific pro- and anti- inflammatory eicosanoids. We found that common LTA4H polymorphisms in human populations control susceptibility to TB and leprosy. People with LTA4H genotypes that result in intermediate levels of inflammation are protected from TB. Intriguingly, individuals with LTA4H genotypes that maximize pro-inflammatory responses fared just as poorly as those with genotypes resulting in inadequate inflammation. These findings suggest an entirely new paradigm in our understanding of TB susceptibility. People are hypersusceptible for two diametrically opposed reasons - an inadequate response to the infection or an overexuberant hyperinflammatory response that is equally detrimental to the host. This proposal will explore the hypothesis that host-directed therapies personalized to common genotypes will provide a fundamentally new approach to combating TB. In strong support of my hypothesis, I have found that human LTA4H genotypes determine clinical responsiveness to standard adjunctive therapy for TB meningitis. We will use the zebrafish model to understand the repercussions of these hypo- and hyperinflammatory states for TB pathogenesis and disease outcome. We will examine drugs that specifically target host eicosanoid pathways as an entirely new avenue of anti-TB treatment. Lastly, the modeling of hypo- and hyperinflammatory states will provide a platform for whole animal, in vivo screening for new drugs that target host eicosanoid pathways. Public Health Relevance: This proposal aims to develop new approaches to treating tuberculosis, a disease that kills nearly two million people annually and is estimated to infect one third of the world's population. The tailoring of effective adjunctive therapies based on patient genotype could have immediate impact on the treatment of TB meningitis, a particularly deadly form of the disease.

Mycobacterium tuberculosis (Mtb) infections kill approximately 1.5 million people annually. Although tuberculosis generally remains confined to the lung, pathogenic mycobacteria also are able to disseminate to other tissues. The interplay of bacterial and host factors that contribute to dissemination is incompletely understood. We identified an outbreak strain that presents with high rates of extrapulmonary dissemination and an extremely high rate of tuberculous bone disease. We have sequenced and assembled the NCG genome and find that is a member of an ancient Mtb lineage. We have identified the Type VII secretion system substrate EsxM as intact in the outbreak strain but truncated in all modern Mtb strains and hypothesize that the ancient variant is an important contributor to bone dissemination. We have developed a zebrafish model to directly examine dissemination and bone disease during mycobacterial infection with Mycobacterium marinum. Using this model we can perform live visualization of osteoblast and osteoclast dynamics. We have found that the full-length versions of EsxM found in M. marinum and ancient lineages of Mtb promote dissemination to bone and are sufficient to modulate macrophage motility. We will 1) interrogate the function of EsxM in modulating the behavior of infected macrophages; 2) identify mechansisms by which EsxM interacts with specific host proteins in macrophages 3) translate these findings in mouse models of dissemination and bone disease. These studies will provide insights into the genetic basis of mycobacterial dissemination and bone disease, a fundamentally important question in our understanding of tuberculosis and other infectious diseases.

Abstract Mycobacterium tuberculosis kills approximately 1.5 million people annually. It has long been observed that mycobacterial granulomas can be extensively vascularized, but the functional consequences of this vascularization have not been fully examined. Using a zebrafish mycobacterial infection model that recapitulates important aspects of human mycobacterial granulomas, we found that granuloma-induced angiogenesis coincides with the generation of local hypoxia and transcriptional induction of the canonical pro- angiogenic molecule Vegfa. Interception of this pathway with clinically used inhibitors resulted in reduced burden and improved outcome. Building on our preliminary data that suggest a role for the VEGF pathway and the bacterial lipid trehalose dimycolate, we will define the cellular and molecular mechanisms by which pathogenic mycobacteria promote the pro-angiogenic environment of mycobacterial granulomas to facilitate their own growth, dissemination and survival. We also will probe the role of a parallel canonical angiogenic signaling pathway ? the angiopoietin/Tie2 pathway ? for which we have identified a novel host-directed drug that we have shown to be effective in reducing mycobacterial burden in zebrafish. Finally, we will build on our recent data showing induction of these pathways in human granuloma specimens and probe the functional effects of human genetic variants in mycobacterial infection. Overall, this proposal tests the hypothesis that, in a striking parallel to tumor biology, the interplay of angiogenesis, hypoxia and compromised vasculature contributes to mycobacterial pathogenesis. Ultimately, the modulation of these pathways may provide new strategies for host- directed therapies for tuberculosis.

Abstract Granulomas form as a conserved host response to a variety of inflammatory and infectious stimuli. As granulomas assemble, macrophages interdigitate and undergo a striking morphological transition, taking on an epithelioid appearance. The basis for this transformation and the consequences for disease are not fully understood. We have identified a conserved reprogramming of macrophages that underlies the assembly and stability of mycobacterial granulomas. Using a zebrafish model, we find that epithelial modules and structures are induced during granuloma formation and are critical for granuloma integrity. In this project we will 1) use long-term intravital imaging to assess key epithelial modules engaged by macrophages during granuloma formation and how disruption of the mycobacterial granuloma can lead to improved host outcome; 2) test the role of the IL-4/IL-13/Stat6 axis during macrophage reprogramming events; 3) based on RNA-seq analysis of macrophages isolated from infected animals, assess the role of the sonic hedgehog (Shh) pathway in granuloma formation, maintenance, and infection trajectory. Findings from this project will be translated into human disease. Overall, this proposal will test the hypothesis that, in a striking parallel to mesenchymal-to- epithelial transitions in cancer, macrophages draw on classical developmental signaling pathways to undergo an epithelial-like transition and construct the central structure of tuberculosis. A new perspective on this critical structure may have important implications for our understanding of disease progression and the design of new therapeutic approaches.