Background: Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils. NETs protect the host against infection by trapping and facilitating phagocytosis of potentially harmful pathogens. Objectives: The aim of the current study was to investigate the effects cigarette smoke condensate (CSC) on phorbol-ester (PMA)-mediated NETosis in vitro, as well as the effects of cigarette. Methods: Isolated human blood neutrophils were exposed to PMA (6.25 ng/ml) in the presence or absence of CSC (40-80 μg/ml) for 90 min at 37oC. Alternatively neutrophils of non-smokers and smokers were activated with PMA (6.25 ng/ml) for 90 min at 37oC. NET formation was measured using a spectrofluorimetric procedure to detect extracellular DNA and fluorescence microscopy was used to visualize nets. Oxygen consumption by PMA-activated neutrophils was measured using an oxygen sensitive electrode. Cotinine levels were measured in smokers and non-smokers for objective confirmation of smoking status Results: Activation of neutrophils with PMA was associated with induction of NETosis that was significantly attenuated in the presence of CSC (40 and 80 μg/ml), with mean fluorescence intensities of 65% and 66% of that observed with untreated cells, respectively, and confirmed by fluorescence microscopy. The rate and magnitude of oxygen consumption by activated neutrophils pre-treated with CSC (80 μg/ml) was significantly less than that observed with untreated cells (73% of the control system), indicative of decreased production of reactive oxidant species in the presence of CSC. When comparing smokers and non- smokers, neutrophils from smokers showed a decrease in both oxygen consumption and the number of NET-forming cells consistent with attenuation of NET formation due to inhalation of cigarette smoke. Conclusion: The inhibition of NETosis observed in the presence of CSC and CS (in smokers) correlated with attenuation of oxygen consumption by PMA-activated neutrophils suggesting a mechanistic relationship between these events. Smoking-related attenuation of NETosis may impair host immune responses and increase the risk of respiratory infections, in vivo. / Dissertation (MSc)--University of Pretoria, 2016. / Immunology / MSc / Unrestricted

<p>Mild-moderate equine asthma (EA) is a commonly encountered disease of racehorses that affects performance. Decreasing dust exposure is crucial in the treatment of equine asthma. Dry hay, because of its high dust content, is known to increase the risk of airway inflammation. Feeding haylage, steamed hay, or hay pellets instead can help to decrease dust exposure. Haylage may also contribute to the resolution of airway inflammation by providing higher levels of omega-3 polyunsaturated fatty acids (Ω-3). Higher levels of Ω-3 are associated with an increase in pro-resolving lipid mediators (PRLM), essential molecules in the resolution of airway inflammation. </p> <p> The studies presented in this dissertation were designed to test the hypothesis that feeding low-dust forages would decrease airway inflammation in racehorses, and that haylage would provide superior resolution of airway inflammation compared to other low dust forages due to changes in systemic Ω-3 and PRLM concentrations associated with increased dietary Ω-3 intake.</p> <p>Three clinical trials were conducted to determine the effect of low-dust forages on airway inflammation. Environmental exposures were measured at the breathing zone, and bronchoalveolar lavage (BAL), and differential cytology counts were performed as measure of airway inflammation. </p> <p>The first clinical trial was a pilot study performed with 7 Standardbred racehorses. Horses were randomly assigned to eat hay (n=3) or haylage (n=4) for 6 weeks while in training. Measurements were performed at baseline and after 2, 4, and 6 weeks. Results showed a decrease in respirable dust, and β-glucan exposure in the horses fed haylage when compared to those fed hay. BAL neutrophil proportion was significantly lower at weeks 2, 4, and 6 when compared to baseline and at week 6 when compared to horses fed hay. </p> <p> The second clinical trial was performed on 19 mild asthmatic horses from the teaching herd. The diet of these horses was changed from dry hay to haylage (n=9) or hay pellets (n=10) for 6 weeks. Measurements were performed at baseline, week 3, and week 6. Results indicated that horses eating haylage and hay pellets were exposed to similar dust levels that were significantly lower than when they were eating dry hay. BAL neutrophil proportion was significantly lower in horses eating haylage when compared to baseline and to horses eating hay pellets at week 6. Horses eating haylage exhibited a significant decrease in stearic acid concentration at week 6. Pro-resolving lipid mediators (Resolvin D1, Resolvin E1, and Lipoxin A₄) did not affect neutrophil apoptosis or efferocytosis <i>in vitro</i>.</p> <p>The third clinical trial was performed on 73 thoroughbred racehorses actively racing and training. Horses were randomly assigned to eat dry hay or to change the forage to steamed hay or haylage. No other change in the management of the horses was allowed. Measurements were performed at baseline (n=73), week 3 (n=69), and week 6 (n=53). Results indicated that respirable dust exposure was significantly reduced when racehorses were fed steamed hay or haylage in place of dry hay. Respirable dust exposure was positively associated with BAL neutrophil proportions. Feeding haylage also significantly decreased exposure to respirable endotoxins. Horses eating haylage for 3 weeks had significantly lower BALF neutrophil proportion when compared with baseline. Also, at week 3, horses eating haylage showed a significant decrease in mast cell proportion, and horses eating steamed hay had a significant decrease in eosinophil proportion. At week 6, horses eating haylage maintained significantly lower BALF neutrophil proportions compared to baseline, and horses eating hay for 6 weeks. Concentration of Ω-3 and PRLM were not increased in horses eating haylage when compared to horses fed hay. Eicosapentaenoic acid was significantly higher on the horses eating haylage when compared to horses eating steamed hay. </p> <p>In conclusion, feeding low-dust forages is sufficient to decrease breathing zone exposure of horses to respirable dust. Despite similar dust exposure, haylage was the only low-dust forage that resulted in resolution of neutrophilic airway inflammation; however, the mechanism remains unclear.</p>

Both of the acute phase reactants, C-reactive protein and fibrinogen, as well as neutrophils have been shown to accumulate at sites of tissue injury or inflammation. The association of C-reactive protein with neutrophils and the concomitant degradation of this ligand by a phorbol 12-myristate 13 acetateactivatable membrane-associated neutral protease has been shown in previous studies. Degradation of C-reactive protein by the neutrophil protease was shown to result in peptides with an ability to modulate various immune functions of the neutrophil. The aim of this study has been to investigate specific characteristics of the protease, with respect to cellular distribution and molecular size. The ability of this neutrophil membrane-associated protease to degrade the acute phase protein, fibrinogen was investigated. The mechanism of degradation of both C-reactive protein and fibrinogen during their association with the neutrophil was also examined. The neutrophil protease, capable of degrading C-reactive protein, was also associated with the cytoskeleton and was proposed to be a submembrane protease localised at sites of attachment of the membrane with the cytoskeleton. The protease was found to have a molecular mass of approximately 600 kDa which, on sodium dodecyl sulphate polyacrylamide gel electrophoresis, separated into four bands which migrated to molecular mass values of 209 kDa, 316 kDa, 398 kDa and 501 kDa. This protease also possessed fibrinogenolytic activity. The fibrinogen degradation products generated by this neutrophil membrane-associated protease were distinct from the products generated by the fibrinogenolytic systems of plasmin, human neutrophil elastase and neutrophil lysosomal enzymes and were unclottable through cleavage of the Aα chain from the N-terminus and the Bβ and γ chains from the C-terminus. N-terminal cleavage of the Aα chain by the neutrophil membrane-associated protease generated the Aα1-21 peptide, previously regarded as a unique consequence of elastase activity. Degradation of C-reactive protein and fibrinogen occurred as a result of their interaction with the neutrophil near to the CD11c integrin receptor. This interaction resulted in the egress of proteolytic activity into the extracellular medium. The fibrinogen products generated outside the cell associated with the neutrophil via the β₂ integrin receptors and the IgG Fc receptor. The interaction of the Creactive protein degradation products with the neutrophil could not be determined. Both C-reactive protein and fibrinogen are degraded by non-stimulated neutrophils but activation with phorbol 12- myristate 13 acetate resulted in maximum degradation This upregulation of activity was achieved through activation of H7 and trifluoperazine inhibitable cellular kinases and changes in microfilament assembly. The generation of non-clottable fibrinogen together with possible modulation of neutrophil receptormediated functions by the fibringen degradation products as well as the knowledge that the neutrophil protease generates C-reactive protein peptides with immunomodulatory activity implicates this neutrophil membrane-associated protease in the modulation of various inflammatory processes.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

Human polymorphonuclear leukocytes (PMN) are a first line of defense against fungal infections. PMN express numerous pattern recognition receptors (PRR) that facilitate identification of invading microorganisms and ultimately promote resolution of disease. Dectin-1 (β-glucan receptor) is a PRR expressed on several cell types and has been studied on monocytes and macrophages. However, the role played by dectin-1 in the recognition and killing of fungi by PMN is unknown. We investigated the ability of dectin-1 to mediate human PMN phagocytosis and fungicidal activity. Dectin-1 was expressed on the surface of PMN from all subjects tested (n=29) and in an intracellular compartment that co-sedimented with azurophilic granules in Percoll density gradients. Soluble β-glucan and mAb GE2 (anti-dectin-1) inhibited binding and phagocytosis of zymosan by human PMN (e.g., ingestionwas inhibited 40.1% by 3O min, p<0.001), and blocked reactive oxygen species production. Notably, soluble β-glucan and GE2 inhibited phagocytosis and killing of Candida albicans by PMN (inhibition of killing was 54.8% for β-glucan and 36.2% for GE2, p<0.01). Our results reveal a mechanism whereby PMN dectin-1 plays a key role in the recognition and killing of fungal pathogens by the innate immune system.

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Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...

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