Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses
Pranvera Sadiku, … , Moira K.B. Whyte, Sarah R. Walmsley
Pranvera Sadiku, … , Moira K.B. Whyte, Sarah R. Walmsley
Published September 1, 2017; First published August 14, 2017
Citation Information: J Clin Invest. 2017;127(9):3407-3420. https://doi.org/10.1172/JCI90848.
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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 2

Phd2-deficient neutrophils display augmented inflammatory responses.

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Phd2-deficient neutrophils display augmented inflammatory responses. (A...
(A–G) Murine inflammatory BAL neutrophils were isolated from WT (white bars) and myeloid-specific Phd2–/– (black bars) mice 24 hours after in vivo LPS (3 mg) challenge and studied either at point of isolation (A–C) or following aging in culture for 6 hours (D–G). (A and D) Chemotaxis. Chemotaxis to KC (100 nM) was determined using neuroprobe chambers. (B and E). Respiratory burst. Change in DCF emission, an indicator of cellular oxidative stress, was quantified by flow following 30 minutes stimulation with fMLP (10 μM). (C and F) Inner mitochondrial membrane potential. FL2 geometric mean fluorescence with tetramethylrhodamine, methyl ester (TMRM) uptake was quantified by flow cytometry in the presence or absence of CCCP (10 μM). CCCP was used as a negative control for the TMRM measure of inner mitochondrial membrane potential and works by collapsing the proton gradient. TMRM is a cell-permeant cationic fluorescent dye that accumulates in active mitochondria and is distributed throughout the cytosol when mitochondrial membrane potential collapses. (G) Mitochondrial ROS. MitoSOX fluorescent emission was quantified by flow cytometry in the presence or absence of CCCP (10 μM). (H) Inflammatory BAL neutrophil bacterial phagocytosis. Alexa Fluor 488–conjugated E. coli BioParticles (MOI 5:1) were administered to inflammatory BAL neutrophil for 1 hour. (I and J) Apoptosis. Peripheral blood neutrophils were isolated from WT (white bars) and knockout Phd2–/– (black bars) mice and cultured in the presence/absence of LPS (100 ng/ml) in normoxia (19 kPa, 21% O2) or hypoxia (3 kPa, 3% O2) for 5 (I) or 9 (J) hours and apoptosis assessed by morphology. (K) Phd2fl/fl MRP8-Cre+/– (MRP8;Phd2fl/fl) and WT control mice were nebulized with LPS (3 mg) and cells harvested by BAL for total cell counts and neutrophil differential counts. Data represent mean ± SEM, n = 4. MFI, mean fluorescence intensity. P values obtained via 2-way ANOVA (A, D, I, J) and unpaired t test (G and K).