Impact of Activation and Airway Transmigration on Human Neutrophil Metabolism Open Access

Patel, Yash (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/m900nv22q?locale=en
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Abstract

Recent studies of dendritic cells and macrophages have shown that these critical regulators of innate and adaptive immunity undergo profound metabolic reprogramming during activation. Neutrophils are the most abundant leukocyte subset in human blood and the first line of defense against pathogens in the body. Prior studies in inflammatory diseases such as cystic fibrosis (CF) suggest that similar metabolic changes are seen upon neutrophil activation to those described in activated dendritic cells and macrophages. Specifically, in the context of CF, neutrophils migrate into the lung lumen in large numbers and display an array of new functions therein, suggesting metabolic licensing, i.e., the ability to use exogenous nutrients to adapt to a new environment. However, metabolic changes in activated neutrophils remain poorly characterized so far, and this study proposes to tackle this current gap in knowledge. The significance of this study is that neutrophils are recognized to play a critical role in the progressive destruction of the CF lungs via pathological reprogramming, and that a better understanding of their metabolic regulation will bring about potential new avenues for therapy. We hypothesize that upon blood neutrophil exposure to pro-inflammatory mediators (PMA, fMLF, LPS, LTB4) there will be an increase in oxygen consumption rate (OCR, reflecting NADPH oxidase activity), and in extracellular acidification rate (ECAR, reflecting extracellular lactate production by glycolysis). Furthermore, we hypothesize that these effects will be further amplified in neutrophils transmigrated in vitro to mimic those found in the lumen of the lung. Understanding the effect of agonists on metabolism of PMNs, the most abundant leukocyte subset in humans, is an ongoing endeavor. Here in this study, we provide proof of concept for the use of the Seahorse bio-analyzer to study the impact of various mediators on PMN metabolism (measured as changes in extracellular acidification rate, ECAR, and oxygen consumption rate, OCR). Also we are able to show pilot data suggesting PMNs have the ability to be further activated after having been submitted to recruitment and activation by CF ASN in the transmigration model which was not the prior understanding.

Table of Contents

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

i. Disease burden of cystic fibrosis (CF).................................1

ii. Genetic basis of CF and pathophysiology............................1

iii. PMN-mediated inflammation in CF....................................2

iv. Metabolic licensing of PMNs.............................................4

v. Study Rationale .............................................................5

II. Materials and Methods............................................................................6

i. Sample collection processing.............................................6

ii. Seahorse energetic bio-analyzer assay...............................7

iii. Transmigration model.....................................................9

III. Results................................................................................................10

IV. Discussion............................................................................................12

V. Figures..................................................................................................14

i. F1.................................................................................14

ii. F2................................................................................15

iii. F3................................................................................16

iv. F4................................................................................17

v. F5.................................................................................18

vi. F6................................................................................19

vii. F7...............................................................................20

viii. F8..............................................................................21

ix. F9................................................................................22

x. F10...............................................................................23

xi. F11..............................................................................24

xii. F12.............................................................................25

xiii. F13............................................................................26

VI. References.........................................................................................27

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