PHD2 Is a Regulator for Glycolytic Reprogramming in Macrophages

Abstract

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1␣ (HIF-1␣) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior.KEYWORDS PDK, prolyl-4-hydroxylase domain, dioxygenases, hypoxia, macrophages M acrophages are an essential component of innate immunity and are well recognized to play critical roles in inflammation, tumor progression, and tissue repair, for example, after an ischemic insult (1). Under aerobic conditions, the oxidative breakdown of pyruvate within the mitochondria is the prevalent source of energy in most cells. Upon a decrease in oxygen availability, cells shift the metabolism toward anaerobic glycolysis. In line with this, macrophages can use aerobic or anaerobic glycolysis for energy production, depending on the context. There is a growing understanding that macrophage function can be altered by cellular metabolism (2). One of the key factors in switching aerobic to anaerobic metabolism at the transcriptional level is the hypoxia-inducible factor (HIF). HIF comprises two subunits: the constitutively regulated HIF␤ subunit and one of three oxygen-regulated HIF␣ subunits (HIF-1␣, HIF-2␣, or HIF-3␣) (3). The protein stability of HIF␣ is regulated by the three prolyl-4-hydroxylase domain (PHD) enzymes, PHD1, -2, and -3, which hydroxylate HIF␣ in an oxygen-dependent manner (for a review, see references 4 and 5). The hydroxylated product is recognized by the pVHL protein, which results in ubiquitination and proteasomal degradation of the ␣-subunit. In hypoxia, the hydroxylation and degra-

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

“…This was associated with a normal Phd2 –/– baseline ( Supplemental Figure 1C ) and LPS-stimulated total white cell and differential cell counts ( Supplemental Figure 1D ) in blood and correlated with enhanced levels of the neutrophil chemokines KC and MIP-1B ( Supplemental Figure 2, A–D ). In keeping with a neutrophil-intrinsic response, Phd2 -deficient airway neutrophils expressed higher levels of CXCR2 and CD11a ( Figure 1, G and H ), with no effect of Phd2 loss on BMDM cytokine expression ( Supplemental Figure 2, E–H ), as previously reported ( 26 ). Importantly, maximal neutrophil recruitment reached equivalence between genotypes at 24 hours, suggesting Phd2 –/– mice had more rapid neutrophil recruitment rather than recruiting greater cell numbers ( Figure 1I ).…”

Effects of cobalt and chromium ions on oxidative stress and energy metabolism in macrophages in vitro

“…Immortalized cells, as opposed to primary cells, were selected to avoid potential inter‐donor variability. However, limitations of the RAW 264.7 macrophage model include its murine origin, as well as higher metabolic rates and OCR than primary murine macrophages …”

Crosstalk Between Hypoxia and ER Stress Response: A Key Regulator of Macrophage Polarization

“…Other oxygen sensors, such as prolylhydroxylases (PHDs) upstream of HIF, also directly contribute to the glycolytic reprogramming of macrophages. To take a specific example, the PHD2 isoform has been found to be important for regulating the glycolytic switch by altering PDK1 activity, as demonstrated by specific PHD2 deletion in the macrophage lineage in vivo (73). The shutdown of the TCA cycle under hypoxic conditions due to the lack of oxygen and to the effects of NO on the mitochondrial ETC causes intermediates, such as citrate and succinate, to accumulate.…”