Characterization of Nucleobindin2/Nesfatin-1 in the Lung and Neutrophils
Date
2018-06-08
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-8764-2449
Type
Thesis
Degree Level
Masters
Abstract
Neutrophil-mediated pulmonary inflammatory conditions such as chronic obstructive pulmonary disease and acute lung injury/adult respiratory distress syndrome are all associated with high mortality rates. While they have been studied extensively, effective treatments remain to be found, suggesting a need to further understand the underlying mechanisms. Nucleobindin 2(NUCB2)/nesfatin-1 is a multifunctional protein originally found to have a role in satiety regulation; it has since been implicated in many other biological processes including inflammation. Studies have shown expression of NUCB2/nesfatin-1 in mouse lungs, and through animal studies, researchers have also suggested it to play an anti-inflammatory and anti-apoptotic role in traumatic brain injury and subarachnoid hemorrhage-induced oxidative brain damage in rats. Cell culture studies, on the other hand, found the addition of nesfatin-1 to chondrocytes to be pro-inflammatory. In addition, cultured chondrocytes and adipocytes showed that NUCB2/nesfatin-1 is upregulated following inflammatory stimulation. Thus far, no studies have characterized the expression of NUCB2/nesfatin-1 in lungs or neutrophils or its role in acute lung inflammation. Hence, the objective of my study was to determine the localization of NUCB2/nesfatin-1 in normal and inflamed lungs and neutrophils, then examine the role it plays following inflammatory stimulus.
In our first study, immunohistochemistry and immune-gold electron microscopy was used to locate NUCB2/nesfatin-1 in normal and inflamed human and mouse lungs and neutrophils. We found that NUCB2/nesfatin-1 localizes in the bronchiolar epithelium, alveolar septa, vascular endothelium, immune cells and red blood cells of normal and inflamed human and mouse lungs; subcellularly, the protein is present in both the nucleus and cytoplasm. Furthermore, localization does not change with inflammatory status. Studies of NUCB2/nesfatin-1 distribution within human neutrophils stimulated with LPS revealed that the protein accumulates within 0.5μm of the plasma membrane after 90mins of stimulation. NUCB2/nesfatin-1 was consistently at higher concentrations in the euchromatin compared to heterochromatin, and cytoplasm compared to nucleus.
Following the localization studies, we performed mechanistic studies into the role of NUCB2/nesfatin-1 during LPS-induced acute lung injury by using wild type (WT) and NUCB2 knock-out mice (NKO) mice. Acute lung injury was accessed through histological evidence of injury, changes in vascular permeability and inflammatory response. Results revealed that NKO lungs had higher adherent neutrophil accumulation, and inflammatory cytokine concentration than WT; vascular permeability was not significantly different between NKO and WT. Further, examining bone marrow-derived mouse neutrophils stimulated with LPS suggested that NKO neutrophils may secrete less inflammatory cytokines compared to WT.
Lastly, we determined that LPS-stimulation does not change NUCB2/nesfatin-1 mRNA or protein expression in mouse lungs. Protein expression of NUCB2/nesfatin-1 in mouse neutrophils were similarly unchanged following LPS treatment.
Taken together, our results suggest that NUCB2/nesfatin-1 is a constitutively expressed protein in WT mice and plays an anti-inflammatory role during acute lung inflammation by inhibiting adherent neutrophil accumulation and inflammatory cytokine expression. Its presence in epithelial, endothelial and immune cells may enable it to inhibit neutrophil infiltration and adhesion, whereas its presence in neutrophilic euchromatin and its apparent ability to regulate inflammatory cytokine expression suggest it may play a role in regulating cytokine transcription.
Description
Keywords
Nesfatin-1, NUCB2, Nesfatin-3, Neutrophils, Lung, Inflammation, Localization, LPS, Human, Mouse
Citation
Degree
Master of Science (M.Sc.)
Department
Veterinary Biomedical Sciences
Program
Veterinary Biomedical Sciences