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Title: Characterisation of the pathophysiological role of chloride ion channels in human leukocytes function
Author: Alothaid, Hani M. M.
ISNI:       0000 0004 7655 435X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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Cystic fibrosis (CF) is a disease that arises due to dysfunction in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, causing thickening of mucus and bacteria colonisation of the airways. The disease has a prevalence of 151 per 100,000 people in the UK. In the airway, CFTR has been shown to form a functional complex with S100A10 – a calcium (Ca2+) binding protein – and annexin A2 – a Ca2+-regulated membrane-binding protein – in a cAMP/PKA-dependent manner to mediate cell secretion. In endothelial cells, activation of calcineurin (CaN)-like phosphatase by cAMP/PKA has been shown to enhance the release of von Willibrand factor (vWF) from Weibel-Palade bodies (WPBs). This study thus aims to establish the extent and importance of regulation of chloride (Cl¯) channels (CFTR and outwardly rectifying Cl¯ ion channel (ORCC)) via cAMP-dependent pathways and LPS in the release of proinflammatory cytokines, phagocytosis of bacteria and survival of phagocytosed bacterial cells by immune cells. We have successfully demonstrated interaction of CaN, S100A10 and AnxA2 in PMAtreated monocytic THP-1 cells in response to cAMP/PKA activation. PKA was also found to regulate Cl¯ channels (CFTR and ORCC) in these cells. In addition, LPS from P. aeruginosa, but not E. coli, was found to induce Cl¯ conductance via CFTR/ORCC in both PMA-treated monocytic THP-1 cells and human peripheral blood monocytes (HPBMs). This Cl¯ transport requires PKA and CaN. Investigation into the role of Cl¯ on phagocytotic ability of the immune cells showed that Cl¯ channels are important for successful phagocytosis and killing of P. aeruginosa by PMA-treated monocytic THP-1 cells and HPBMs-derived macrophages (MDMs). In addition, LPS induced the release of both IL-1ß and TNF-a from HPBMs. However, PKA/CaN appears not to be involved in CFTR/ORCC-mediated cytokine release. Further investigation showed that while PKA/CaN is not required for CFTR-mediated inflammatory responses and the phagocytotic ability of immune cells, Ca2+ and EPAC2 are absolutely required in a PKAindependent manner. In conclusion, LPS from P. aeruginosa activates CFTR/ORCC, and this is facilitated by PKA/CaN activity. Moreover, EPAC2 and Ca2+ may be the requisite factors for CFTR/ORCC-mediated inflammatory response to occur in immune cells.
Supervisor: Muimo, Richmond ; Robson, Louise Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available