Spatial immunophenotyping of bacilli and secreted virulence factors in tuberculosis granulomas

Oregon Health & Science University, Portland OR

Investigators

Abstract

PROJECT SUMMARY Tuberculosis (TB) is caused by the pathogen Mycobacterium tuberculosis (Mtb) and its arsenal of secreted virulence factors, exhibiting one of the largest global disease burdens caused by a single infectious agent. TB is primarily a pulmonary disease with the latent form that affects 90% of infected individuals being asymptomatic, while the active form is characterized by a spectrum of clinical findings ranging from mild to severe, including cachexia, malaise, fever, and sputum production. A classic finding in the lungs of TB patients is the granuloma, a microenvironment of host immune cells recruited to infection foci. While the granuloma is believed to be host- protective by containing bacteria, concentrating immune responses, and preventing dissemination, granulomas may be detrimental as a fibrotic niche that is impermeable to T cells, favorable for Mtb to lie dormant, and impenetrable to antibiotics. These ineffective granulomas may contribute to latent TB in which true bacterial sterilization is indiscernible, leaving the disease incurable and patients at risk for activation. The host-pathogen interactions occurring within granulomas are thus important mechanistic targets for therapies but remain elusive. While previous granuloma histopathology has resulted in extensive descriptions of host microenvironment, the role of individual Mtb-expressed virulence factors in shaping these phenotypes has been underappreciated. This proposal aims to stain granulomas from non-human primate (NHP) lungs and human lymph nodes with an extensive panel of Mtb antigen-specific probes (i.e. commercial antibodies, novel in-house nanobody tetramers, acid-fast staining, and RNAScope) and advanced host multiplexing techniques (i.e. Phenocycler) to elucidate the localization, abundance, and molecular influence of Mtb factors on the granulomatous immune response and its success. Preliminary data show that Mtb bacilli are phenotypically heterogenous in terms of virulence factor expression, may localize far beyond the myeloid core where most bacilli reside, and secrete factors into host cells across the infected lung. The central hypothesis is that Mtb differentially regulates its virulence factor repertoire to manipulate immune dynamics, facilitating favorable granulomas that promote bacterial survival. This fellowship proposal also outlines a training plan customized for refining a rigorous physician-scientist in lung pathology and emerging/neglected infectious diseases. This includes an exceptional mentorship team with expertise across histopathology and TB, experimental NHP cohorts, nanobodies, and other reagents/resources unique to Oregon Health and Science University, and a training experience at the SAMRC Centre for TB Research in endemic South Africa. Completion of this proposal will result in 1) understanding Mtb heterogeneity in the infected lung, which may contribute to ineffective therapies and latent TB, 2) elucidating the role of virulence factors in granuloma progression, and 3) identifying novel Mtb-host interactions as therapeutic targets.