Evaluation of neutrophil extracellular trap production in pregnancy
Introduction During pregnancy there is a unique problem for the maternal immune system in supporting the development of a non-allogenic foetus whilst maintaining an effective immune response to invading pathogens. This is usually achieved through the development of an anti-inflammatory immune p...
| Main Author: | |
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2017
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| Online Access: | https://eprints.nottingham.ac.uk/42855/ |
| _version_ | 1848796585468624896 |
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| author | Tarbox, Rebecca Clare |
| author_facet | Tarbox, Rebecca Clare |
| author_sort | Tarbox, Rebecca Clare |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Introduction
During pregnancy there is a unique problem for the maternal immune system in supporting the development of a non-allogenic foetus whilst maintaining an effective immune response to invading pathogens. This is usually achieved through the development of an anti-inflammatory immune phenotype. A novel mechanism of bacterial killing, the formation of neutrophil extracellular traps (NETs), is characterised by the release of an extracellular mesh of DNA, histones and granule proteins. However, this process also leads to local endothelial and epithelial damage, through exposure to NET components, resulting in inflammation. In 40% of cases of spontaneous preterm birth (sPTB), bacterial infection has been implicated and whether NETs play a role in the development of this syndrome is unknown. The aim of this study was to determine whether leukocytes from umbilical cord blood (CBL) and maternal blood (MBL) produced NETs and whether this process is altered in the presence of infection or preterm pre-labour rupture of membranes (PPROM).
Methods
CBL or MBL or were isolated from whole blood by dextran sedimentation. NET production was induced by stimulation with lipopolysaccharide (LPS), a polymer contained within the cell wall of gram-negative bacteria, Lipoteichoic acid (LTA) and peptidoglycan (PGN), both polymers contained within the cell wall of gram-positive bacteria. Phorbol 12-myristate-13-acetate (PMA), is a widely studied inducer of NET formation and was used as a positive control. NET production was measured by a cell impermeable SYTOX® green assay. In CBL this was supplemented with immunofluorescence to determine the presence and localisation of neutrophil elastase (NE) and the digestion of NET structures with micrococcal nuclease (MNase), with NE concentrations in NET fragments determined.
Results
All of the ligands used stimulated the externalisation of DNA from CBL and MBL in a concentration dependent manner (p<0.0001). NET production was confirmed in CBL by visualisation of NE in extracellular structures emitting from the leukocytes. In CBL, there is increased NET formation with PGN stimulation where a maternal GBS infection is present than seen in controls (p<0.0001). In MBL from PPROM in the 2nd trimester, the NET response was heightened when compared with MBL at term with LTA, PGN and PMA stimulation (p<0.05). Comparisons made between CBL and MBL revealed that CBL are more sensitive to PMA stimulation (p=0.0032), whereas MBL are more sensitive to PGN stimulation (p=0.0392).
Discussion
Our observations demonstrate that NET formation occurs in CBL and MBL in response to LPS, LTA, PGN and PMA. Further, the level of NET production is altered with GBS infection (PGN) and PPROM (LTA, PGN and PMA). During pregnancy, NETs form part of the host-defence mechanism to infection and dysregulation of this process may be a novel component of sPTB. |
| first_indexed | 2025-11-14T19:50:19Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-42855 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T19:50:19Z |
| publishDate | 2017 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-428552025-02-28T13:46:18Z https://eprints.nottingham.ac.uk/42855/ Evaluation of neutrophil extracellular trap production in pregnancy Tarbox, Rebecca Clare Introduction During pregnancy there is a unique problem for the maternal immune system in supporting the development of a non-allogenic foetus whilst maintaining an effective immune response to invading pathogens. This is usually achieved through the development of an anti-inflammatory immune phenotype. A novel mechanism of bacterial killing, the formation of neutrophil extracellular traps (NETs), is characterised by the release of an extracellular mesh of DNA, histones and granule proteins. However, this process also leads to local endothelial and epithelial damage, through exposure to NET components, resulting in inflammation. In 40% of cases of spontaneous preterm birth (sPTB), bacterial infection has been implicated and whether NETs play a role in the development of this syndrome is unknown. The aim of this study was to determine whether leukocytes from umbilical cord blood (CBL) and maternal blood (MBL) produced NETs and whether this process is altered in the presence of infection or preterm pre-labour rupture of membranes (PPROM). Methods CBL or MBL or were isolated from whole blood by dextran sedimentation. NET production was induced by stimulation with lipopolysaccharide (LPS), a polymer contained within the cell wall of gram-negative bacteria, Lipoteichoic acid (LTA) and peptidoglycan (PGN), both polymers contained within the cell wall of gram-positive bacteria. Phorbol 12-myristate-13-acetate (PMA), is a widely studied inducer of NET formation and was used as a positive control. NET production was measured by a cell impermeable SYTOX® green assay. In CBL this was supplemented with immunofluorescence to determine the presence and localisation of neutrophil elastase (NE) and the digestion of NET structures with micrococcal nuclease (MNase), with NE concentrations in NET fragments determined. Results All of the ligands used stimulated the externalisation of DNA from CBL and MBL in a concentration dependent manner (p<0.0001). NET production was confirmed in CBL by visualisation of NE in extracellular structures emitting from the leukocytes. In CBL, there is increased NET formation with PGN stimulation where a maternal GBS infection is present than seen in controls (p<0.0001). In MBL from PPROM in the 2nd trimester, the NET response was heightened when compared with MBL at term with LTA, PGN and PMA stimulation (p<0.05). Comparisons made between CBL and MBL revealed that CBL are more sensitive to PMA stimulation (p=0.0032), whereas MBL are more sensitive to PGN stimulation (p=0.0392). Discussion Our observations demonstrate that NET formation occurs in CBL and MBL in response to LPS, LTA, PGN and PMA. Further, the level of NET production is altered with GBS infection (PGN) and PPROM (LTA, PGN and PMA). During pregnancy, NETs form part of the host-defence mechanism to infection and dysregulation of this process may be a novel component of sPTB. 2017-07-14 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/42855/1/Rebecca%20Tarbox%20-%20Evaluation%20of%20Neutrophil%20Extracellular%20Trap%20Production%20in%20Pregnancy.pdf Tarbox, Rebecca Clare (2017) Evaluation of neutrophil extracellular trap production in pregnancy. MRes thesis, University of Nottingham. Neutrophils Extracellular matrix Innate immunity Leukocytes Infection Preterm pre-labour rupture of membranes |
| spellingShingle | Neutrophils Extracellular matrix Innate immunity Leukocytes Infection Preterm pre-labour rupture of membranes Tarbox, Rebecca Clare Evaluation of neutrophil extracellular trap production in pregnancy |
| title | Evaluation of neutrophil extracellular trap production in pregnancy |
| title_full | Evaluation of neutrophil extracellular trap production in pregnancy |
| title_fullStr | Evaluation of neutrophil extracellular trap production in pregnancy |
| title_full_unstemmed | Evaluation of neutrophil extracellular trap production in pregnancy |
| title_short | Evaluation of neutrophil extracellular trap production in pregnancy |
| title_sort | evaluation of neutrophil extracellular trap production in pregnancy |
| topic | Neutrophils Extracellular matrix Innate immunity Leukocytes Infection Preterm pre-labour rupture of membranes |
| url | https://eprints.nottingham.ac.uk/42855/ |