Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.758286
Title: Cellular metabolism and the NLRP3 inflammasome as therapeutic targets in cystic fibrosis
Author: Scambler, Thomas
ISNI:       0000 0004 7431 060X
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CFTR protein, an anion channel that conducts Cl- and HCO3- and regulates amiloride-sensitive Na+ channels. In both animal and in vitro models, CFTR dysfunction results in excessive neutrophilic inflammation, Na+ absorption and inflammasome activation, with a strong interleukin (IL)-1 signature. This study’s hypothesis is that CF is associated with a pathogenic increase in Na+ transport, which occurs due to the loss of CFTR-dependent inhibition of amiloride-sensitive Na+ channels and drives excessive inflammation and metabolism at the molecular and systemic level. Primary human monocytes, macrophages and bronchial epithelial cell lines, with characterised CF-associated mutations, were cultured in vitro and stimulated for NLRP3 inflammasome activation. Differentiated macrophages were characterised using flow cytometry. Cytokines, ion fluxes and metabolites were measured from in vitro stimulations using colorimetric and fluorometric assays. Patients with CF were observed to have a systemic proinflammatory cytokine signature accompanied by an in vitro hyperactivation of the NLRP3 inflammasome and metabolic pathways associated with proinflammatory immune phenotypes. Excessive Na+ influx in cells with CF-associated mutations, which created a propensity for excessive K+ efflux and cellular energy demand upon stimulation, was observed in this study. Primary CF monocytes and epithelial cell lines hyper-responded to NLRP3 stimulation with disproportionate IL-1b and IL-18 secretion, inhibited by pre-treatment with small molecule and peptide inhibitors of Na+ channels, metabolic pathways and the NLRP3 inflammasome. In addition, a small cohort of patients with CF-associated mutations on a 3-month study of small molecule CFTR modulator therapy showed a reduction in inflammation and glycolysis. This is the first study to demonstrate a link between excess Na+ absorption, a characteristic intrinsic feature of CF, and increased inflammation and metabolism. Future therapies will need to focus on rectifying Na+ absorption if they are to obtain maximal therapeutic benefit. Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CFTR protein, an anion channel that conducts Cl- and HCO3- and regulates amiloride-sensitive Na+ channels. In both animal and in vitro models, CFTR dysfunction results in excessive neutrophilic inflammation, Na+ absorption and inflammasome activation, with a strong interleukin (IL)-1 signature. This study’s hypothesis is that CF is associated with a pathogenic increase in Na+ transport, which occurs due to the loss of CFTR-dependent inhibition of amiloride-sensitive Na+ channels and drives excessive inflammation and metabolism at the molecular and systemic level. Primary human monocytes, macrophages and bronchial epithelial cell lines, with characterised CF-associated mutations, were cultured in vitro and stimulated for NLRP3 inflammasome activation. Differentiated macrophages were characterised using flow cytometry. Cytokines, ion fluxes and metabolites were measured from in vitro stimulations using colorimetric and fluorometric assays. Patients with CF were observed to have a systemic proinflammatory cytokine signature accompanied by an in vitro hyperactivation of the NLRP3 inflammasome and metabolic pathways associated with proinflammatory immune phenotypes. Excessive Na+ influx in cells with CF-associated mutations, which created a propensity for excessive K+ efflux and cellular energy demand upon stimulation, was observed in this study. Primary CF monocytes and epithelial cell lines hyper-responded to NLRP3 stimulation with disproportionate IL-1b and IL-18 secretion, inhibited by pre-treatment with small molecule and peptide inhibitors of Na+ channels, metabolic pathways and the NLRP3 inflammasome. In addition, a small cohort of patients with CF-associated mutations on a 3-month study of small molecule CFTR modulator therapy showed a reduction in inflammation and glycolysis. This is the first study to demonstrate a link between excess Na+ absorption, a characteristic intrinsic feature of CF, and increased inflammation and metabolism. Future therapies will need to focus on rectifying Na+ absorption if they are to obtain maximal therapeutic benefit. Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CFTR protein, an anion channel that conducts Cl- and HCO3- and regulates amiloride-sensitive Na+ channels. In both animal and in vitro models, CFTR dysfunction results in excessive neutrophilic inflammation, Na+ absorption and inflammasome activation, with a strong interleukin (IL)-1 signature. This study’s hypothesis is that CF is associated with a pathogenic increase in Na+ transport, which occurs due to the loss of CFTR-dependent inhibition of amiloride-sensitive Na+ channels and drives excessive inflammation and metabolism at the molecular and systemic level. Primary human monocytes, macrophages and bronchial epithelial cell lines, with characterised CF-associated mutations, were cultured in vitro and stimulated for NLRP3 inflammasome activation. Differentiated macrophages were characterised using flow cytometry. Cytokines, ion fluxes and metabolites were measured from in vitro stimulations using colorimetric and fluorometric assays. Patients with CF were observed to have a systemic proinflammatory cytokine signature accompanied by an in vitro hyperactivation of the NLRP3 inflammasome and metabolic pathways associated with proinflammatory immune phenotypes. Excessive Na+ influx in cells with CF-associated mutations, which created a propensity for excessive K+ efflux and cellular energy demand upon stimulation, was observed in this study. Primary CF monocytes and epithelial cell lines hyper-responded to NLRP3 stimulation with disproportionate IL-1b and IL-18 secretion, inhibited by pre-treatment with small molecule and peptide inhibitors of Na+ channels, metabolic pathways and the NLRP3 inflammasome. In addition, a small cohort of patients with CF-associated mutations on a 3-month study of small molecule CFTR modulator therapy showed a reduction in inflammation and glycolysis. This is the first study to demonstrate a link between excess Na+ absorption, a characteristic intrinsic feature of CF, and increased inflammation and metabolism. Future therapies will need to focus on rectifying Na+ absorption if they are to obtain maximal therapeutic benefit.
Supervisor: McDermott, Michael ; Peckham, Daniel ; Savic, Sinisa Sponsor: University of Leeds
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.758286  DOI: Not available
Share: