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

Innate immune cells serve as the first line of defense by detecting pathogens through pathogen- and damage-associated molecular patterns (PAMPs and DAMPs). Recognition of PAMPs triggers the synthesis and secretion of pro-inflammatory proteins, with the inflammasome playing a key role in the secretion of interleukin-1β (IL-1β), a crucial part of the innate immune response. Upon inflammasome activation, the release of signal-peptide lacking cytoplasmic proteins such as IL-1β occurs through non-classical pathways known as unconventional protein secretion (UCPS), which vary depending on the cell type and stimulus which helps tailor the extent of pro-inflammatory protein secretion. Broadly, UCPS cargoes are released either via direct release through plasma membrane pores, or by entering vesicular intermediates that fuse with the plasma membrane. The aim of this thesis was to establish cell line models to study these distinct secretory pathways. We characterized the secretion pathway responsible for IL-1β release from neutrophil-like differentiated HL-60 (dHL-60) cells and macrophage-like THP-1 cells, as primary neutrophils and macrophages are known to utilize vesicular intermediates and plasma membrane pores, respectively, for IL-1β release. Our findings revealed that upon NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, dHL-60 cells—unlike parental HL-60 cells—secreted IL-1β in a NLRP3-dependent manner, independent of pyroptosis. IL-1β secretion in dHL-60 cells was unaffected by autophagy inhibition, unlike primary neutrophils. Furthermore, treatment with brefeldin A (BFA), a classical secretion inhibitor, significantly reduced IL-1β release in dHL-60s. Examination of IL-1β synthesis and secretion following treating with BFA indicated that BFA affects multiple stages, including synthesis and release of IL-1β. In contrast, THP-1 macrophage-like cells showed robust IL-1β secretion that was not affected by BFA treatment, consistent with a GSDMD pore-mediated unconventional secretion route. These findings indicate that dHL-60 and THP-1 cells release IL-1β via distinct secretory pathways. Additionally, we evaluated available online tools for prediction of UCPS cargoes. Here, we found that these tools have low accuracy, highlighting the importance of developing improved prediction models which could be built by studying UCPS in cell line models established here

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