The effect of extracellular acidosis on tissue destruction in tuberculosis
File(s)
Author(s)
Whittington, Ashley Michael
Type
Thesis
Abstract
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.
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.
Version
Open Access
Date Issued
2017-09
Date Awarded
2018-01
Advisor
Friedland, Jon
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)