Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Mitchell, Thomas C.

Committee Co-Chair (if applicable)

Uriarte, Silvia M.

Committee Member

Shirwan, Haval

Committee Member

Mcleish, Kenneth

Committee Member

Rane, Madhavi

Committee Member

Kosiewicz, Michele

Author's Keywords

TLR4; neutrophils; MPLA; accessory cells; survival; CF

Abstract

Neutrophils respond to bacterial LPS through Toll-like receptor 4 (TLR4), which activates or potentiates immune defensive functions and prolongs cell survival. Activation of TLR4 signaling in neutrophils is beneficial for effective clearance of LPS-bearing Gram-negative bacteria, but may also drive aberrant inflammation if not stringently regulated. The regulatory processes by which neutrophil functions are calibrated to respond appropriately to different LPS-bearing bacteria are incompletely understood. Described here are investigations that reveal an unexpected sensitivity of TLR4 in neutrophils to small changes in LPS structure typical of various Gram-negative bacteria, including those that are dangerously virulent (Escherichia coli and Salmonella enterica), opportunistically pathogenic (Pseudomonas aeruginosa), or beneficial inhabitants of healthy tissue (commensal bacteria). TLR4 signal strength varies with heterogeneity in the extent of phosphorylation and acylation of lipid A, the active center of LPS. Neutrophils respond vigorously to fully phosphorylated lipid A structures typical of virulent bacteria, but were surprisingly refractory to monophosphorylated variants of lipid A. This pattern may reflect the low threat posed by commensal bacteria, many of which are naturally monophosphorylated, and helps explain the low endotoxicity of a therapeutically useful vaccine adjuvant manufactured from LPS through chemical dephosphorylation (Chapter II). TLR4-mediated effects on neutrophil survival were found to be responsive to changes in cellular microenvironment such as growth in suspension or as adherent cells, in the presence or absence of non-neutrophilic accessory cells. A loss of intrinsic TLR4 survival signaling in adherent neutrophils relative to those cultured in suspension was discovered, and has important implications as a checkpoint at which neutrophil abundance can be controlled through withdrawal of survival factors (Chapter III). Unchecked neutrophil accumulation is a serious problem for cystic fibrosis (CF) disease patients chronically infected with P. aeruginosa (PA). Tests of neutrophil responses to a lipid A variant that is seen only in severe, late stages of CF were conducted for the first time. The variant lipid A, with a unique hepta-acylated structure, robustly primed neutrophil respiratory bursts, stimulated high levels of IL-8 secretion and prolonged neutrophil survival, but failed to stimulate neutrophil granule exocytosis This pattern is consistent with late stage CF disease in which PA is paradoxically not cleared despite provoking highly inflammatory influxes of activated neutrophils (Chapter IV). Stringent regulation of neutrophil survival and function is necessary for health and occurs in part through the precision with which TLR4 detects structural variants of lipid A, successfully for some Gram-negative bacteria but as an exploited host vulnerability by others.

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