Functional and transcriptional adaptations of blood monocytes recruited to the cystic fibrosis airway microenvironment in vitro 公开

Abstract

Cystic Fibrosis (CF) is an autosomal recessive genetic disease, impacting an estimated 80,000 individuals worldwide, and still without cure. This disease disproportionately affects people of Caucasian descent with over 1,800 confirmed mutations divided into 6 classes impacting the structure and localization of the CF Transmembrane Conductance Regulator (CFTR) ion channel. The dysfunction of CFTR leads to a variety of systemic effects, with the manifestation of lung disease, derived from a pathological triad of obstruction, inflammation, and infection, representing the primary cause of death in patients with CF.

 

CF lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior studies, our group showed that blood neutrophils migrated through the well-differentiated lung epithelium into CF airway fluid supernatant (ASN, purified from patient sputum) in vitro mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. In this study, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment into the lung lumen and exposure to CFASN. We tested this hypothesis by isolating primary human monocytes from blood and transmigrating them in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profiling by RNA sequencing were quantified post-transmigration.

 

Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they, too, fail to protect CF airways from infection.

 

 

Table of Contents

Chapter 1: Introduction to monocyte development, phenotype, function, and relationship to cystic fibrosis airway disease ......................................................................................................................1

Overview of monocyte development and function ............................................................................... 1

Tissue migration and differentiation ..................................................................................................... 4

Monocytes: “of mice and men” .............................................................................................................. 6

Monocyte transcriptional profile ........................................................................................................... 7

Transcripts involved in monocyte-to-macrophage differentiation ..................................................... 8

Monocyte phagocytosis and clearance of pathogens .......................................................................... 11

Monocyte scavenging ............................................................................................................................ 12

Overview of cystic fibrosis .................................................................................................................... 14

CF airway disease pathogenesis ........................................................................................................... 15

CFTR effects on monocyte/macrophage function .............................................................................. 18

Models of CF disease ............................................................................................................................ 19

Chapter 2: Monocyte phenotype and function after recruitment to CF airways ............................. 21

Introduction ........................................................................................................................................... 22

Methods and Materials ......................................................................................................................... 23

Results .................................................................................................................................................... 25

Discussion .............................................................................................................................................. 31

Chapter 3: Monocyte transcriptional adaptation after recruitment to cystic fibrosis airways......... 33

Introduction ........................................................................................................................................... 34

Methods and Materials ......................................................................................................................... 35

Results .................................................................................................................................................... 37

Discussion .............................................................................................................................................. 44

Chapter 4: Conclusions and perspectives ........................................................................................ 46

References ...................................................................................................................................... 51

Abbreviations ................................................................................................................................. 64

Figures Index .................................................................................................................................. 67

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology
关键词	cystic fibrosis in vitro immunology monocytes airway transmigration
Committee Chair / Thesis Advisor	Dr. Rabin Tirouvanziam, Emory University
Committee Members	Dr. Jake Kohlmeier, Emory University Dr. Larry Boise, Emory University Dr. Sean Stowell, Harvard University Dr. Larry Anderson, Emory University

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