Cellular Signaling Mechanisms Underlying the Angiogenic Response to Mycobacterial Infection

Pathological angiogenesis is a widespread biological phenomenon that influences the progression of various diseases, including autoimmune conditions, cancers, and microbial infections. One infection in particular, tuberculosis, is associated with the induction of a potent pro-angiogenic signaling cascade that facilitates bacterial growth and accelerates disease progression. A synthesis of early studies on bacterial factors that drive host angiogenesis with modern genetic findings identified the mycobacterial glycolipid trehalose 6-6'-dimycolate (TDM) as a critical factor driving vascular endothelial growth factor (VEGFA) production and angiogenesis during mycobacterial infection. Despite these recent findings, many of the underlying host response mechanisms remain unknown. The introductory chapter will serve to introduce the reader to the major concepts addressed in this work: Mycobacterium tuberculosis and the disease it causes, the role of macrophages in health and disease, the function of pattern recognition receptors in detecting microbial ligands, the specific downstream intracellular signaling pathway of interest for this work (mediated by the transcription factor, nuclear factor of activated T cells, NFAT), the contributions of angiogenesis to diverse contexts and pathologies, and the promise of host-directed therapies to overcome challenges associated with traditional treatment approaches in infectious disease.

Chapter 2 describes the new and existing methodological approaches that were required to complete this work. This work utilizes the zebrafish-Mycobacterium marinum model of tuberculosis infection to facilitate in depth in vivo observation and quantitation of these phenomena. Using this model in tandem with human macrophage cell culture, I was able to model major aspects of the host-pathogen interface, enabling me to identify a critical role for a macrophage-C-type lectin receptor-NFATC2-VEGFA signaling axis required for the angiogenic response to mycobacterial infection and TDM, findings that comprise the core of this work and are detailed at length in Chapter 3.

The analysis of the large amounts of data generated in this work required creative approaches to data processing and analysis. To this end, I have developed a set of novel processing modalities in Python and R that are capable of the rapid and reproducible processing of images as well as certain aspects of automated data collection therefrom. These macros, many written for the FIJI/ImageJ programming environment, serve as the infrastructure on which the rest of this work has been built. These will be detailed in Chapter 4.

Finally, this body of work leaves many questions as yet unanswered. While it is clear that NFAT signaling is required for VEGFA production, the precise mechanism by which this may work is unclear and could be mediated by either direct DNA binding or indirect activation or cooperative binding with some other transcriptional activator. There also exist a variety of other potential NFAT- and angiogenesis-related phenotypes worthy of exploring using the tools and approaches I have developed. It is my hope that the findings herein stimulate further study on the contributions of NFAT signaling to the host immune response to mycobacterial infection and evaluation of the potential of NFAT inhibition as host-directed therapy to tuberculosis.