Differential roles for the oxygen sensing enzymes PHD1 and PHD3 in the regulation of neutrophil metabolism and function

Background Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. Methods We used a combination of in vivo and ex vivo models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. Results We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter ex vivo neutrophil phenotypes or in vivo outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. Conclusions PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target

Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS

Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS

Author correction : Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation

In the version of this article originally published, in the Methods section "Mouse LPS ALI model," the second sentence needed clarification of wording and dosage (mg kg–1, not mg g–1) and has been amended to read "Mice were treated daily (days 1–4 post-LPS), by subcutaneous injection, with PBS or 0.75 mg kg–1 of porcine CSF-1 fused to the Fc region of porcine IgG1a (generated by David Hume), prior to cull on day 5" in the HTML and PDF versions of the article

Investigating the metabolic plasticity of inflammatory neutrophils during infection

Neutrophils are required to function and survive under challenging conditions characterised by tissue hypoxia and nutrient scarcity. Neutrophils are well suited to rapidly respond to infection and engage with metabolic pathways for the rapid generation of substrates used to meet their energy demands, reactive oxygen species (ROS) production, and delaying constitutive apoptosis programmes. By using a variety of assays, I investigated the dynamic metabolic adaptations of infected neutrophils exposed to both hypoxia and glucose depletion. I observed that glucose depletion reduces apoptosis of infected neutrophils, and that hypoxia further promotes neutrophil phagocytosis. Mass spectrometry metabolite quantification and metabolic flux analysis revealed that neutrophils upregulate the pentose phosphate pathway to sustain ROS production and bacterial killing. Gluconeogenesis, glycogenolysis and the glycerol 3-phosphate pathways are all critical for neutrophil bacterial control and hypoxic adaptation. Finally, investigation of PHD3-/- neutrophils revealed a phenotype of improved bacterial control and reduced inflammation which might have beneficial therapeutic implication

Neutrophil HIF-1α stabilisation is augmented by mitochondrial ROS produced via the glycerol 3-phosphate shuttle

Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signaling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilization of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarized mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be an important consideration when looking for treatments of inflammatory conditions in which HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity