Inflammasome-Mediated Interleukin-1β Drives Neutrophilic Airway Inflammation in Cystic Fibrosis 公开

Rao, Sanjana K. (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/c247ds851?locale=zh
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Abstract

Cystic fibrosis (CF) patients experience progressive airway destruction due to opportunistic pathogen colonization and neutrophil-dominated chronic inflammation. In order to investigate the inflammatory response promoting disease progression, we assessed neutrophil inflammasomes and the resulting activation of the IL-1 (interleukin-1) pathway, consisting of pro-inflammatory IL-1β signaling and an endogenous inhibitor, interleukin-1 receptor antagonist (IL-1RA). Specifically, we performed ex vivo assays of CF human blood and airway samples with comparison to healthy control (HC) subjects and in vitro neutrophil inflammasome stimulations. Further, we assessed bioactivity of IL-1β to identify the potential role of IL-1RA in modulating the IL-1β activatory cascade, and to investigate potential correlations with disease severity. Results show that IL-1β production is significantly elevated in CF, compared to HC and chronic obstructive pulmonary disease (COPD) controls, confirming that CF disease is linked with IL-1 signaling. Furthermore, we observed a strong correlation between neutrophil numbers and IL-1β levels, consistent with a pro-inflammatory loop of neutrophil recruitment and IL-1β secretion that itself recruits more neutrophils. Results from inflammasome stimulation and follow-up in vitro assays together demonstrate that IL-1β secretion may not be concomitant with cell death, contrary to conventional belief. Finally, using a cell line genetically engineered to detect bioactive IL-1β, we found that IL-1β in CF airway samples is indeed bioactive and that this bioactivity is closely regulated by IL-1RA. Ultimately, results gathered strongly suggest that IL-1β is a clinically relevant modulator of CF, and a potential target for therapeutic intervention.

Table of Contents

Table of Contents

I. Introduction.........................................................................1

i. Background ...........................................................1

ii. Genetic basis of CF .................................................1

iii. Pathophysiology of CF airway disease.......................2

iv. PMN-mediated inflammation....................................2

v. Inflammasome activation.........................................3

vi. Modulation of IL-1β pathway....................................4

vii. Study rationale.......................................................5

II. Materials and Methods........................................................5

i. Human subjects......................................................5

ii. Sample collection processing....................................6

iii. ELISA..................................................................6

iv. Human Embryonic Kidney (HEK)-Blue Cells...............8

v. in vitro inflammasome activation.............................10

vi. LDH Assay...........................................................11

vii. Statistical analysis...............................................12

III. Results............................................................................12

IV. Discussion........................................................................14

V. Tables and Figures.............................................................18

i. T1...................................................................18

ii. F1...................................................................18

iii. F2...................................................................19

iv. F3...................................................................20

v. F4...................................................................21

vi. F5...................................................................22

vii. F6...................................................................23

viii. F7...................................................................24

ix. F8...................................................................25

x. F9..................................................................26

xi. F10................................................................27

xii. F11................................................................28

xiii. F12................................................................29

xiv. F13................................................................31

VI. References......................................................................32

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