Date of Award

Fall 8-22-2025

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

First Advisor

Prabhodh Abbineni

Abstract

The innate immune system is the first line of defense against pathogens. Innate immune cells, such as macrophages and neutrophils, recognize pathogens through the recognition of pathogen associated molecular patterns or damage associated molecular patterns (PAMPS & DAMPS). When a pathogen is recognized, innate immune cells will start clearing the pathogen and secreting pro-inflammatory cytokines, such as interleukin-1β (IL-1β) to initiate the innate immune response. Release of IL-1β occurs through an unconventional secretory pathway triggered by inflammasome activation. Activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome cleaves both pro-IL-1β into mature IL-1β, and full length gasdermin-D (GSDMD) into a pore-forming N-terminal fragment (NT-GSDMD). In macrophages, the secretion of IL-1β is mediated through NT-GSDMD pores that form on the plasma membrane. Plasma membrane NT-GSDMD pores also allow the influx of water leading to cell lysis in a process called pyroptosis. In macrophages, pyroptosis occurs rapidly after inflammasome activation due to the immediate localization of NT-GSDMD to the plasma membrane. However, in neutrophils, pyroptosis is not a rapid process, rather they seem to be resistant to pyroptosis even under the same inflammasome activation conditions as macrophages. Previous studies have shown that NT-GSDMD localizes to intracellular vesicles in neutrophils directly after inflammasome activation and not the plasma membrane. The regulation of pyroptosis and NT-GSDMD subcellular localization is poorly characterized in neutrophils. To investigate the basis of neutrophil pyroptosis resistance and NT-GSDMD trafficking, we first identified a genetically tractable cell line model that mimics the pyroptosis phenotype observed in primary neutrophils. We determined that the HL60 cell line, when differentiated into neutrophil-like cells, recapitulates the pyroptosis-resistance phenotype observed in primary neutrophils upon NLRP3 inflammasome activation. We then employed pharmacological inhibition of secretory pathways and microscopy-based localization studies to characterize NT-GSDMD localization and trafficking in differentiated HL60 cells. We also established a doxycycline-inducible, flag-tagged NT-GSDMD expressing HeLa cell line, to investigate NT-GSDMD trafficking in a reductionist setting independent of upstream inflammasome activation. These complementary cell line tools now allow for dissection of NT-GSDMD localization and pyroptosis regulation. Determining how neutrophils regulate NT-GSDMD localization and resist lytic death may reveal novel approaches of therapeutic intervention for controlling neutrophil-driven inflammation in pathological contexts.

Download

Share

COinS