Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses
Pranvera Sadiku, Joseph A. Willson, Rebecca S. Dickinson, Fiona Murphy, Alison J. Harris, Amy Lewis, David Sammut, Ananda S. Mirchandani, Eilise Ryan, Emily R. Watts, A.A. Roger Thompson, Helen M. Marriott, David H. Dockrell, Cormac T. Taylor, Martin Schneider, Patrick H. Maxwell, Edwin R. Chilvers, Massimilliano Mazzone, Veronica Moral, Chris W. Pugh, Peter J. Ratcliffe, Christopher J. Schofield, Bart Ghesquiere, Peter Carmeliet, Moira K.B. Whyte, Sarah R. Walmsley
Pranvera Sadiku, Joseph A. Willson, Rebecca S. Dickinson, Fiona Murphy, Alison J. Harris, Amy Lewis, David Sammut, Ananda S. Mirchandani, Eilise Ryan, Emily R. Watts, A.A. Roger Thompson, Helen M. Marriott, David H. Dockrell, Cormac T. Taylor, Martin Schneider, Patrick H. Maxwell, Edwin R. Chilvers, Massimilliano Mazzone, Veronica Moral, Chris W. Pugh, Peter J. Ratcliffe, Christopher J. Schofield, Bart Ghesquiere, Peter Carmeliet, Moira K.B. Whyte, Sarah R. Walmsley
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Research Article Inflammation Metabolism

Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses

Abstract

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF–prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.

Authors

Pranvera Sadiku, Joseph A. Willson, Rebecca S. Dickinson, Fiona Murphy, Alison J. Harris, Amy Lewis, David Sammut, Ananda S. Mirchandani, Eilise Ryan, Emily R. Watts, A.A. Roger Thompson, Helen M. Marriott, David H. Dockrell, Cormac T. Taylor, Martin Schneider, Patrick H. Maxwell, Edwin R. Chilvers, Massimilliano Mazzone, Veronica Moral, Chris W. Pugh, Peter J. Ratcliffe, Christopher J. Schofield, Bart Ghesquiere, Peter Carmeliet, Moira K.B. Whyte, Sarah R. Walmsley

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Figure 4

Inflammatory neutrophils deficient in Phd2 display enhanced glycolytic capacity and glycogen storage with parallel increases in ATP production and utilization.

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Inflammatory neutrophils deficient in Phd2 display enhanced glycolytic c...
Murine inflammatory BAL neutrophils from WT (white bars) and Phd2–/– (black bars) mice were studied following isolation 24 hours after in vivo challenge with nebulized LPS (3 mg). (A) Glycolytic metabolites. Murine inflammatory BAL neutrophils were cultured in the presence of U-13C glucose for 6 hours under conditions of normoxia (21% O2) and hypoxia (3% O2). Incorporation into G6P and redistribution of 13C carbons derived from U-13C glucose were measured using LC-MS. Data are presented as relative metabolite abundance and show mean ± SEM, n = 3. A diagram of the glycolytic and gluconeogenesis pathways depicting 2 possible outcomes resulting in the generation of G6P m3 isotopomer by recycling of 13C carbons is included. Total G6P is a measure of both unlabeled 12C G6P and all other mass isotopomers of G6P denoted m0–m6, where m0 contains only 12C carbons and m6 with all 6 carbons 13C. (B) Glycogen content. WT (white bars) and myeloid-specific Phd2–/– (black bars) inflammatory BAL neutrophils were lysed at time of isolation and following 6 hours culture in normoxia or hypoxia and intracellular glycogen stores quantified by a colorimetric assay. Data represent mean ± SEM, n = 5. (C–F) Energetics. BAL neutrophils were cultured for 6 hours ex vivo and relative ATP (C), ADP (D), and AMP (E) levels determined by LC-MS, enabling calculation of energy charge (F). Data represent mean ± SEM, n = 3. DHAP, dihydroxyacetone phosphate. P values obtained via unpaired t test.