Browsing by Author "Tobin, David M"

Background

Methods

Results

Conclusions

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.

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.

Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis, which kills more people worldwide than any other infectious disease. In 2017, nearly 2 million people died of tuberculosis. Despite the advent of antibiotics targeting Mtb, the global spread of tuberculosis continues. The development of antibiotic resistance within the bacteria has further complicated the already long and difficult course of treatment for the disease. New therapeutics are necessary to combat tuberculosis. A novel treatment strategy is the use of host-directed therapies, which provide an orthogonal approach to killing intracellular pathogens. Rather than directly targeting bacterial pathways, which may lead to the development of mutations that result in resistance to the drug, host directed therapies (HDTs) target the host immune response to the disease. To uncover these host directed therapies, we have utilized the zebrafish-Mycobacterium marinum model system. Using zebrafish infected with their natural pathogen, Mycobacterium marinum, a close genetic relative to Mtb, we show that we can enhance the ability of the host immune response to kill intracellular bacteria.

In Chapter 1, I introduce tuberculosis as a disease and discuss the past, present and future of treating the disease. I discuss potential host targets for immune modulating therapies, including autophagy, inflammation, and inflammasomes. I highlight the role of calcium signaling in immune cells, specifically neutrophils and macrophages. I briefly describe zebrafish as a model system, emphasizing their use to study immune responses and host-pathogen interactions. In Chapter 2, we show calcium is required for immune cell activity and motility in neutrophils. Calcium is a signal that leads neutrophils not only to wound sites but also to sites of infection and inflammation. We then enhance calcium signaling through potentiation of the membrane channel P2RX7 with the small molecule clemastine, an FDA-approved over-the-counter antihistamine in Chapter 3. We show that clemastine treatment reduces bacterial burden in a P2RX7 –dependent manner in zebrafish larvae. P2RX7 activation leads to assembly of inflammasomes in macrophages, a key immune cell of mycobacterial infection. In human mycobacterial disease, many of the bacteria are contained within structures called granulomas, in which host macrophages and other immune cells have formed a cuff around the bacteria, creating a space that is recalcitrant to treatment with frontline antibiotics. Clemastine is effective in these established infection structures, indicating that it may be a feasible strategy to treat human tuberculosis. We discuss how mycobacteria evade the host immune response and demonstrate how a small molecule can overcome these evasion strategies for improved host outcome.

In the past 200 years, tuberculosis (TB) has caused more deaths than any other infectious disease and currently infects more people than it has at any other time in human history. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is an obligate human pathogen that has evolved through the millennia to become the archetypal human-adapted pathogen. This work focuses on the evolutionary framework by which Mtb emerged as a specialized human pathogen, and applying this perspective to outbreak strains and the strategies Mtb deploys to manipulate its host environment.

There are seven major lineages that define the human-adapted species of Mtb. Beijing lineage strains, also known as Lineage 2 strains, have emerged as important drivers of global Mtb burden due to the elevated rates of drug-resistance, rapid disease progression, and increased transmission characteristics they display. Beijing lineage strains are endemic to East Asia, but have recently expanded globally. In Chapter 2, we investigate circulating Beijing strains in Guatemala, a country with limited data regarding the molecular epidemiology of Mtb and few reported cases of disease caused by Beijing strains. We report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

Pairing whole genome sequencing with outbreak strains displaying unusual disease phenotypes provides compelling insight into the genetic changes underlying the corresponding disease presentation. Mtb most commonly causes lung disease, but can also disseminate to other tissue sites. We identified an outbreak strain of Mtb that presented clinically with unusually high rates of extrapulmonary and bone disease in seemingly immunocompetent individuals. We find that the outbreak was caused by an ancient strain of Mtb that, like other ancestral strains and animal-adapted mycobacterial pathogens, carries a full-length version of the Type VII secreted effector EsxM. Here we show that EsxM is required for the full disseminative properties of mycobacterium-infected macrophages and is sufficient to directly enhance macrophage motility. Moreover, we find that EsxM has been inactivated in all modern strains of Mtb, suggesting a potential selective advantage to limiting dissemination as Mtb adopted and adapted to its modern pulmonary niche.

Pathogenic mycobacteria have long been known to utilize an array of effectors to manipulate their host environment. Mycobacterial infection initiates the assembly of granulomas, which are discrete host structures composed of tightly associated immune cell aggregates. Macrophages within the granuloma have been described as "epithelioid" due to the morphological transformation they undergo as they interdigitate with their neighboring cells. The epithelial transformation macrophages undergo is central to the formation of the granuloma, yet this transformation has not been well characterized at the molecular level during mycobacterial infection. Our laboratory has utilized the zebrafish to dissect the epithelial transformation of macrophages during mycobacterial infection. In Chapter 4, I outline the transcriptional reprogramming that occurs in granuloma macrophages that underlies this transformation. We find that granulomas exhibit distinct transcriptional profiles from macrophages, and, surprisingly, that immune cells deploy developmental programs to construct the granuloma.

Tuberculous granulomas – the central immune structures of tuberculosis – are the primary site of host-pathogen interactions. These are complex, multi-cellular host immune structures that dictate disease progression and physiology. Due to the heterogenous nature of the cells that contribute the granuloma, signaling cues from diverse populations of cells can be overlooked by bulk analyses. While macrophages and monocytes make up the majority of cells in a given granuloma, other myeloid cells, lymphocytes, and stromal are known to play important roles in infection trajectory and control. A hallmark of tuberculous granulomas are epithelioid macrophages. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition or whether small populations of cells can determine cell signaling at the level of a granuloma.Using the zebrafish-Mycobacterium marinum model of infection together with single-cell RNA-seq analysis of experimentally accessible and genetically tractable zebrafish granulomas we identified 1) the basis of granuloma macrophage transformation and 2) a novel granuloma-associated fibroblast cell. Even in the presence of robust type 1 immune responses that are typical for tuberculous granulomas, countervailing type 2 signals associate with macrophage epithelialization. We find that stat6-mediated type 2 immune signaling is required for macrophage epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell-autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the granuloma structure of mycobacterial infection. In addition, we analyzed wildtype adult mycobacterial granulomas and granulomas from the genetically susceptible lta4h mutant with single-cell resolution in zebrafish. lta4h is implicated in vertebrate control of mycobacterial infection as well as human TB severity. We find that eicosanoid perturbations disrupt the formation of a fibroblast cell layer at the granuloma periphery, which is associated with containment of infection and conserved in human TB granulomas. This unique fibroblast population is marked by a transcriptional profile with mesenchymal and stem-like properties of dermal fibroblasts, including expression of the aldehyde dehydrogenase aldh1a3 and apolipoprotein D. The expression of apod in these cells is associated with ROS regulation in the granuloma. Diverse tuberculous granulomas across patients and tissues in humans also generate these unusual fibroblast populations. These findings establish that crosstalk between stromal and immune cells, shaped by host lipid mediators, contributes to divergent pathological outcomes during mycobacterial infection.

Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of lipid accumulation using the zebrafish-M. marinum model of infection. We find that neutral lipids accumulate cell autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Reducing available free cholesterol and neutral lipids during early infection via treatment with ezetimibe, an FDA-approved drug, resulted in a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell autonomously at early timepoints of mycobacterial infection, and this early lipid accumulation confers a growth advantage to infecting mycobacteria.

This accumulation represents a perturbation of the normal homeostatic mechanisms by which intracellular lipids are tightly controlled. Under homeostatic conditions macrophages are central to the function of returning excess lipids to the liver where they are processed for excretion via the digestive tract. This function of macrophages, termed reverse cholesterol transport, results from the uptake of excess extracellular lipids, followed by the coordinated efflux of these lipids through the transport proteins ABCA1 and ABCG1 and packaging into HDL particles containing ApoAI and ApoAII. This process is controlled via the action of nuclear receptors including PPARγ, PPAR-α, and LXRα.

We performed RNA-seq analysis of gene expression in macrophages both uninfected and infected with mycobacteria and observed a profound down-regulation of all the major lipoproteins. The HDL associated lipoproteins ApoAI and ApoAII were the most profoundly down regulated. Based on this observation we sought to investigate the role of nuclear receptors involved in intracellular lipid sensing and control of apolipoprotein expression. We determined that agonism and antagonism of LXRα signaling decrease and increase infection burden during in vivo studies respectively. We further found that the ApoAI agonizing fibrate drugs fenofibrate and gemfibrozil reduce mycobacterial infection in vivo. This work demonstrates that lipid lowering agents already approved for use in humans might function as relevant adjuvant therapies toward treatment of mycobacterial infections.