Chemical tools to dissect macrophage signaling in inflammation and infection [electronic resource]

Macrophages are immune cells that inhabit almost every tissue of the body and work to maintain organismal homeostasis. They recognize a broad range of deviations from normal (e.g., metabolic imbalance, presence of pathogens, tissue damage) and tailor their responses to return the body to normal. Macrophages are remarkably plastic in their signaling state, which allows them to respond to homeostatic disruptions in a rapid and appropriate manner. Due to this rapid adaptation to new environments, the ability to perturb and observe macrophage signaling in a temporally defined manner is key. Chemical tools allow such rapid manipulation and observation and, in this thesis, I will describe our use of chemical tools such as small molecule inhibitors and optical probes to dissect dynamic macrophage signaling. One mechanism through which macrophages respond to danger is by initiating the formation of protein complexes called inflammasomes. These complexes facilitate activation of the pro-inflammatory cytokines IL-1β and IL-18 and rapid pyroptotic cell death. This causes inflammation, the goal of which is to identify and combat the source(s) of danger. Inflammasome signaling is beneficial for fighting pathogenic bacteria, fungi, and viruses, but aberrant or excessive inflammasome signaling can damage tissues. Chapters 2 and 4 describe the application of small molecule inhibitors to dissect the cellular signals that lead to formation of a specific inflammasome, the NLRP3 inflammasome, which is implicated in the response to bacterial pathogens and is also overactive in metabolic and genetic autoinflammatory disease. Our work in Chapter 2 describes the identification of a small molecule activator of the NLRP3 inflammasome. By characterizing the mechanism of action of this small molecule, we found that disruption of glycolytic metabolism drives NLRP3 inflammasome formation and pyroptotic cell death in macrophages. We find that this mechanism of NLRP3 inflammasome activation is used by host macrophages to sense and react to the intracellular bacterial pathogen Salmonella typhimurium. In Chapter 4, we discuss the development of a live-cell imaging-based screening method that enables comprehensive identification of small molecules that modulate NLRP3 inflammasome formation and pyroptotic cell death. The goals of this project are to further knowledge of cellular signals leading to NLRP3 activation and to identify therapeutic leads for combatting aberrant NLRP3-driven inflammation. Just as macrophages have evolved many ways of recognizing pathogens, pathogens have also evolved many methods of evading detection and can even successfully colonize and replicate within macrophages. For example, Salmonella typhimurium lives in specialized endosomes and lysosomes in host macrophages. In Chapter 3, we describe the development of optical probes that we use to characterize the interplay of endolysosomal Salmonella with host macrophages' endolysosomal enzymes. We focus on a specific family of enzymes, the cysteine cathepsins, because they fulfill anti-bacterial and pro-bacterial functions depending on their localization. We find that upon initial colonization of macrophages, cathepsins can access Salmonella in early endosome-like compartments. In contrast, at later time-points after colonization, Salmonella cause an elevation in endolysosomal pH that impairs cathepsin protease activity and prevents cathepsins from accessing their replicative niche. Taken together, these data convey a complex and changing equilibrium between a bacterial pathogen and the cells that it colonizes.

Keywords: Chemical and Systems Biology.
Advisor: Bogyo Matthew
Meyer Tobias advisor. Thesis advisor
Mochly-Rosen Daria advisor. Thesis advisor
Monack Denise M. advisor. Thesis advisor