Regulation of Leukocyte Inflammation by Small Particles Including Extracellular Vesicles and Viruses Open Access
Dobosh, Brian (Fall 2022)
Abstract
Innate immune cells, which include polymorphonuclear neutrophils (PMNs) and monocytes, are constantly patrolling tissues such as the lung, in search of damaged or dying cells. This dissertation describes two cases in which innate immune cells fail to resolve airway stress and thus promote disease.
In patients with cystic fibrosis (CF), the main cause of morbidity and mortality is lung disease, which is associated with an early, massive and chronic recruitment of PMNs into the airway lumen. Therein, PMNs release potent effector proteins such as neutrophil elastase, myeloperoxidase, interleukin (IL)-1b and IL-18, which can damage the airways. Such pathological PMNs, which we have dubbed “GRIM” (for granule releasing, immunomodulatory, and metabolically active) are observed not only in the airways of CF patients, but also in those of patients with chronic obstructive pulmonary disease (COPD), asthma, and severe coronavirus disease of 2019 (COVID-19). Although PMNs are professional phagocytes, they actively restrict killing of bacteria in CF airways, leading to chronic infections. To date, it is unclear what factors cause PMNs to become GRIM. Work presented here show that extracellular vesicles (EVs) released by GRIM airway PMNs cause naïve, newly immigrated airway PMNs to take on the same fate. More precisely, GRIM PMN-derived EVs carrying the long non-coding RNA MALAT1 cause expression the enzyme HDAC11 in naïve PMNs, which drives them to degranulate, reduce their bacterial killing capacity, and release a new wave of MALAT1+ EVs that perpetuates the pathological cycle. Inhibition or knockdown of HDAC11 or MALAT1 reverses the GRIM phenotype and promotes killing of bacteria.
COVID-19 patients are generally asymptomatic during initial severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication, but may suffer severe immunopathology after the virus has receded and blood monocytes have infiltrated the airways. Patients that lack an early PMN response tend to have a monocyte response that results in cytokine release syndrome and severe -COVID-19. Using an in vitro model, we show that airway-infiltrating monocytes acquire SARS-CoV-2 from infected epithelium and upregulate expression and secretion of inflammatory mediators including CXCL8 and IL-1b, mirroring in vivo data. Next, we characterized viral burden, gene expression and inflammatory mediator secretion by lung epithelial cells and infiltrating monocytes in the absence or presence of the antiviral remdesivir and/or immunomodulatory drug baricitinib. As expected, remdesivir decreased viral burden in both the epithelium and monocytes, while baricitinib enhanced antiviral signaling and decreased specific inflammatory mediators in monocytes. Combined use of baricitinib and remdesivir enhanced the rate of virus clearance from SARS-CoV-2-positive monocytes.
Taken together, when monocytes and PMNs have a discordant immune response to stress, runaway inflammation and lung damage may occur. We have developed methods to interrogate and modulate in vitro how these cells behave in the context of various stressors including virus and extracellular vesicles and have initiated testing of novel treatment approaches including small molecule drugs and DNA- and RNA-based therapeutics to tackle these problems.
Table of Contents
Table of Contents
Chapter 1: INTRODUCTION TO EV BIOLOGY
EV NOMENCLATURE
BIOLOGICAL EFFECTS OF EVS
EV CARGO
EV Carrier Effects
EV Transfer Across Kingdoms
RNA Sorting
EV ANALYSIS BY FLOW CYTOMETRY
Comparison with other methods
Population-based Analysis
Coincident Events
Optics and Hardware
REFERENCES
Chapter 2: INTRODUCTION TO NEUTROPHIL BIOLOGY
NEUTROPHIL DEVELOPMENT IN THE BONE MARROW
CLEARANCE OF PMNS
PMN ACTIVATION
PMNS IN CF LUNG DISEASE
ASSOCIATION OF NE AND OTHER EFFECTOR MOLECULES WITH EVS
IN VITRO MODELING OF PMN EV SIGNALING
THERAPEUTIC POTENTIAL OF EV-BASED APPROACHES
REFERENCES
Chapter 3: NEUTROPHIL-DERIVED EXTRACELLULAR VESICLES PROMOTE FEED-FORWARD INFLAMMASOME SIGNALING IN CYSTIC FIBROSIS AIRWAYS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Supplementary Methods
RESULTS AND DISCUSSION
FIGURES
REFERENCES
Chapter 4: EXTRACELLULAR VESICLE lncRNA MALAT1 DRIVES HDAC11 DEPENDENT CHRONIC INFLAMMATION IN AIRWAY NEUTROPHILS
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
REFERENCES
Chapter 5: BARICITINIB ATTENUATES THE PROINFLAMMATORY PHASE OF COVID-19 DRIVEN BY LUNG-INFILTRATING MONOCYTES
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
SUPPLEMENTARY FIGURES
REFERENCES
Chapter 6: SUMMARY AND FUTURE DIRECTIONS
MUSINGS ON COMMUNICATION VIA EVS
FUTURE DIRECTIONS REGARDING PMN-DRIVEN INFLAMMATION
HDAC11 and MALAT1
Potential Mechanisms Regulating HDAC11
Metabolism and HDAC11
HERVs
REFERENCES
ABBREVIATIONS
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