|dc.description.abstract||Cystic fibrosis (CF) is a genetic disease with pathology highly focused on pulmonary complications. Infection and colonisation of CF airways by well established pathogens (e.g., Pseudomonas aeruginosa, Staphylococcus aureus) are continual issues, and these infections are commonly managed through prolonged use of multiple antibiotics. Neutrophils respond to pulmonary bacterial infections. In CF, however, altered airway physiology and neutrophil function contribute to inflammation, tissue remodeling, and fibrosis. It has been established that circulating neutrophils from individuals with CF are functionally altered, either intrinsically or from the inflammatory environment of the airways, and this alteration appears to involve increased neutrophil survival and reduced apoptosis. Furthermore, this alteration in neutrophil physiology has been related to undefined mediators present within plasma from individuals with CF. Therefore, to better understand disease pathogenesis and improve CF patient management, it is important to identify the plasma factors causing these alterations of the CF neutrophil. With these concerns in mind, the primary objective of this research was to identify plasma factors that altered the physiology of circulating neutrophils in CF, and to determine which neutrophil signaling pathways were stimulated by these factors. A further objective was to investigate how these plasma factors and altered neutrophil functions related to disease progression and medical chart data for individuals with CF.
To address my research objectives, blood samples were collected from 22 adult volunteers with CF who were ΔF508 homozygotes, and from 11 age and sex matched non-CF control volunteers. This study did not incorporate inclusion or exclusion criteria based on clinical assessment of stability for the volunteers with CF. Plasma samples were first screened for the capacity to stimulate the NF-κB pathway using the THP1-Dual cell, NF-κB reporter assay. In addition, proteins present in a plasma sample collected from a volunteer with CF were analysed using mass spectrometry. Plasma samples were also analysed for specific factors known to have stimulatory effects on neutrophils. These factors included G-CSF and CXCL1, which were measured using enzyme-linked immunosorbent assay (ELISA) methodology, and endotoxin, which was measured using the limulus amebocyte lysate (LAL) endotoxin assay. Neutrophils from these blood samples were also isolated and the number of neutrophils isolated/ml of blood were calculated. Some of these circulating neutrophil samples were analysed for gene expression of several pro-inflammatory factors using reverse transcription - quantitative real-time polymerase chain reaction (RT-qPCR). In addition, expression of the anti-apoptotic protein XIAP and phosphorylated XIAP were evaluated through western blotting analysis of neutrophil lysates. Finally, intracellular signaling in circulating neutrophil samples was analysed using kinome arrays and the resulting data was subjected to pathway over-representation analyses. Medical chart data (including detection of sputum bacteria, antibiotic therapy regimen, and lung function testing) was abstracted for 20 of the volunteers with CF and used to perform statistical analyses with laboratory data.
There was a significant (p ≤ 0.01) increase in the numbers of circulating neutrophils isolated/ml blood (i.e., neutrophil numbers) from volunteers with CF when compared to the control group. Furthermore, for the volunteers with CF, significantly (p ≤ 0.01) increased neutrophil numbers were associated with antibiotic regimens that contained the β-lactam antibiotic aztreonam lysine for inhalation (AZLI) (i.e., AZLI positive). Among those volunteers who were AZLI negative, significantly (p ≤ 0.05) decreased circulating neutrophil numbers were associated with volunteers who had S. aureus detected in their sputum (i.e., S. aureus positive). In addition, statistically there was an interaction between the associations of AZLI and S. aureus status with circulating neutrophil numbers, which was indicated by the greatest mean neutrophil numbers being observed in volunteers who were both AZLI positive and S. aureus positive. Volunteers with CF who were AZLI positive also had a significant (p ≤ 0.05) association with decreased lung function parameters FVC % and FEV1/FVC, and trended (p = 0.056) toward a decrease in FEV1 % when compared to those volunteers who were AZLI negative. Similar associations to those made for AZLI were not observed for other antibiotics investigated, including inhaled tobramycin.
When cytokines were analysed in plasma samples from volunteers, it was observed that G-CSF and CXCL1 concentrations were detected in the majority of samples from volunteers with CF. It was also identified among the volunteers with CF who were S. aureus negative, that increased plasma concentrations of G-CSF and CXCL1 were significantly (p ≤ 0.05) associated with volunteers who were AZLI positive. Plasma G-CSF and CXCL1 were also significantly (r(19) = .45, p = .039) positively correlated with each other for the volunteers with CF. In addition, G-CSF concentrations were significantly (r(19) = .53, p = .013) positively correlated with plasma endotoxin levels. CXCL1 concentrations, however, were only significantly (r(5) = .83, p = .024) positively correlated with endotoxin levels among volunteers who were on antibiotic regimens that contained ciprofloxacin, which is a common therapy during CF pulmonary exacerbations. Correlation analysis of plasma cytokines and circulating neutrophil numbers revealed that G-CSF concentrations trended (r(19) = .41, p = .063) toward a positive correlation with neutrophil numbers, and G-CSF-related pathway signaling was altered in the majority of CF neutrophil samples. Alteration of G-CSF-related signaling pathways was further linked to alterations in apoptosis signaling through the “Apoptosis” pathway. Accordingly, western blot analyses revealed that elevated XIAP protein may have been present in CF circulating neutrophils, and this elevated XIAP protein may have been associated with increased G-CSF, endotoxin, and CXCL1 concentrations in plasma. In addition, kinome array analysis revealed significantly (p ≤ 0.05) increased phosphorylation of peptide XIAP S87 among the volunteers with CF who did not have detectable levels of plasma endotoxin (i.e., endotoxin-negative) when compared to the endotoxin negative control volunteers. This increase in phosphorylation of peptide XIAP S87 coincided with alterations in the G-CSF-related signaling pathways “JAK STAT pathway and regulation” (4.4% coverage by array peptides with significantly [p ≤ 0.05] associated phosphorylation levels) and “EPO signaling pathway” (6.6% coverage by array peptides with significantly [p ≤ 0.05] associated phosphorylation levels) as well as alterations in the “Apoptosis” pathway (11.4% coverage by array peptides with significantly [p ≤ 0.05] associated phosphorylation levels) between the endotoxin negative CF and control groups. Furthermore, peptide XIAP S87 was the most informative single peptide on the kinome array. Peptide XIAP S87 was observed to be significantly (p ≤ 0.05) differentially phosphorylated in several of the analyses of kinome array data against parameters from laboratory experiments and medical chart abstracts. For the majority of volunteers with CF, this data strongly implicated XIAP as a key factor involved in the alteration of apoptosis-related pathway signaling in neutrophils. That said, similar results for apoptosis-related pathway signaling and phosphorylation of array peptide XIAP S87 were not observed for volunteers with CF who were endotoxin positive (i.e., who had detectable levels of plasma endotoxin).
Plasma CXCL1 concentrations for volunteers with CF were not correlated (r(19) = -.22, p = .349) with neutrophil numbers, while plasma endotoxin levels were significantly (r(19) = .52, p = .017) positively correlated with neutrophil numbers. Possibly confirming the biological activity of plasma endotoxin, volunteers with CF who were endotoxin positive were observed to have altered neutrophil signaling in the TLR4-related pathway “MyD88 cascade initiated on plasma membrane” (9.6% coverage by array peptides with significantly [p ≤ 0.05] associated phosphorylation levels) when compared to volunteers with CF who were endotoxin negative. For neutrophil samples from volunteers with CF, it was also identified that alteration of the “Apoptosis” pathway may have been associated (10.2% coverage by array peptides with significantly [p ≤ 0.05] associated phosphorylation levels) with volunteer who were S. aureus positive, and that alteration of this pathway did not coincide with alteration of the G-CSF-related signaling pathways for these volunteers. Also, as expected, circulating neutrophil numbers in the volunteers with CF were significantly negatively correlated with parameters of lung function: FEV1 % (r(18) = -.52, p = .019); FVC % (r(18) = -.55, p = .012); and SpO2 (r(18) = -.57, p = .008).
In conclusion, for the volunteers with CF, AZLI was highly associated with neutrophil numbers. Theoretically this association may have been mediated through a release of endotoxin which then stimulated production of CXCL1 and G-CSF. It cannot be excluded, however, that this association with AZLI was driven by disease severity, where increased bacterial burden and lower lung function may have been related to the previously mentioned release of endotoxin and cytokine production. G-CSF may have then stimulated the release of neutrophils from the bone marrow, as it is known to do, and may have also directly activated circulating neutrophils through NF-κB. This activation by G-CSF may have then increased expression and phosphorylation of XIAP, which was probably also influenced by other effectors (e.g., endotoxin, S. aureus). Collectively these effects on the inhibition of apoptosis-related pathway signaling may theoretically explain a mechanism by which reduced apoptosis occurs in circulating neutrophils in relation to plasma from individuals with CF. It is certainly possible that increased numbers of these functionally altered neutrophils were related to disease progression in volunteers with CF, as neutrophil numbers were associated with decreased lung function. Inhaled tobramycin was not observed to have the same associations as AZLI; however, because this was predominantly an exploratory study, it is difficult to ascertain practical meaning from this discrepancy between inhaled antibiotics. In summary, our findings provide evidence that support antibiotic use and pulmonary bacteria being associated with the functional alterations detected in CF circulating neutrophils, which appear to occur through endotoxin and G-CSF-related increases in circulating neutrophil numbers as well as inhibition of apoptosis-related pathway signaling.||