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Title: The effect of extracellular acidosis on tissue destruction in tuberculosis
Author: Whittington, Ashley Michael
ISNI:       0000 0004 8504 4722
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Introduction Mycobacterium tuberculosis (M.tb) infection of macrophages causes the secretion of Matrix Metalloproteinases (MMPs) which degrade the lung extracellular matrix producing tissue destruction and morbidity. Proinflammatory cytokine secretion by macrophages, particularly TNF-α, is critical for M.tb killing and drives further MMP secretion by normal human bronchial epithelial cells (NHBEs). Activated macrophages release lactic acid which lowers extracellular tissue pH and raises lactate levels at sites of infection. Extracellular acidosis is detected by pH-sensing G-protein coupled receptors (TDAG-8, OGR-1 and GPR4). This study investigates how lesional extracellular lactic acidosis modulates TB immune responses. Methods RNA-seq was performed on primary human monocyte-derived macrophages (MDMs) infected with M.tb H37Rv at pH 7.4 or in acidosis (pH 7.0). MDMs were infected with M.tb at varying pH and MMP and cytokine secretion measured by ELISA and intracellular signalling assessed by Western Blot. Acidosis receptor expression was measured by qPCR and Immunohistochemistry of TB lesions. NHBEs were stimulated with TB dependent cytokine networks at varying pH and MMP secretion and acidosis receptor expression assessed. Results RNA-seq identified key genes and pathways regulated by M.tb. Acidosis upregulated extracellular matrix degradation pathways due to increases in MMP expression. In vitro experiments confirmed acidosis increases MMP-1 and -3 from M.tb infected MDMs and decreases TNF-α. This was dependent upon TDAG-8 signalling, the most significantly expressed acidosis sensing GPCR in MDMs. TDAG-8 was highly expressed in granulomas from TB patients. Lactate acting intracellularly also increases MMP-1 secretion by MDMs in TB. NHBEs stimulated with TB dependent cytokine networks increase secretion of MMP-1 and -9 with acidosis, dependent upon OGR-1 signalling. Extracellular lactate further increases NHBE MMP-1 and -9 secretion. Discussion Extracellular acidosis amplifies MMP secretion in TB favouring tissue destruction, whilst decreasing key cytokines that control infection. Acidosis receptors therefore represent novel targets for host directed therapy in Tuberculosis.
Supervisor: Friedland, Jon Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral