Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions.

Oxygen (O2) tension plays a key role in tissue function and pathophysiology. O2-controlled cell culture, in which the O2 concentration in an incubator's gas phase is controlled, is an indispensable tool to study the role of O2 in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O2 tension that cells experience (i.e., pericellular O2). We discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0.0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O2 tensions, we developed a reaction-diffusion model that predicts pericellular O2 tension a priori. This model revealed that the effect of cellular O2 consumption is greatest in smaller volume culture vessels (e.g., 96-well plate). By controlling pericellular O2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A, CD73, NDUFA4L2, etc. and lower levels of HIF1A, CA9, VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O2 tension in cell culture incubators is insufficient to control O2 in cell culture and introduce the concept of pericellular O2-controlled cell culture.

Targeting hypoxia-inducible factor-1 alpha suppresses Helicobacter pylori-induced gastric injury via attenuation of both cag-mediated microbial virulence and proinflammatory host responses.

Helicobacter pylori-induced inflammation is the strongest known risk factor for gastric adenocarcinoma. Hypoxia-inducible factor-1 (HIF-1α) is a key transcriptional regulator of immunity and carcinogenesis. To examine the role of this mediator within the context of H. pylori-induced injury, we first demonstrated that HIF-1α levels were significantly increased in parallel with the severity of gastric lesions in humans. In interventional studies targeting HIF-1α, H. pylori-infected mice were treated ± dimethyloxalylglycine (DMOG), a prolyl hydroxylase inhibitor that stabilizes HIF-1α. H. pylori significantly increased proinflammatory chemokines/cytokines and inflammation in vehicle-treated mice; however, this was significantly attenuated in DMOG-treated mice. DMOG treatment also significantly decreased function of the H. pylori type IV secretion system (T4SS) in vivo and significantly reduced T4SS-mediated NF-κB activation and IL-8 induction in vitro. These results suggest that prolyl hydroxylase inhibition protects against H. pylori-mediated pathologic responses, and is mediated, in part, via attenuation of H. pylori cag-mediated virulence and suppression of host proinflammatory responses.

SUMOylation indirectly suppresses activity of the HIF-1α pathway in intestinal epithelial cells.

The hypoxia-inducible factor (HIF) is a master regulator of the cellular transcriptional response to hypoxia. While the oxygen-sensitive regulation of HIF-1α subunit stability via the ubiquitin-proteasome pathway has been well described, less is known about how other oxygen-independent post-translational modifications impact the HIF pathway. SUMOylation, the attachment of SUMO (small ubiquitin-like modifier) proteins to a target protein, regulates the HIF pathway, although the impact of SUMO on HIF activity remains controversial. Here, we examined the effects of SUMOylation on the expression pattern of HIF-1α in response to pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) in intestinal epithelial cells. We evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression and inhibition of SUMOylation using a novel selective inhibitor of the SUMO pathway, TAK-981, on the sensitivity of HIF-1α in Caco-2 intestinal epithelial cells. Our findings demonstrate that treatment with TAK-981 decreases global SUMO-1 and SUMO-2/3 modification and enhances HIF-1α protein levels, whereas SUMO-1 and SUMO-2/3 overexpression results in decreased HIF-1α protein levels in response to DMOG. Reporter assay analysis demonstrates reduced HIF-1α transcriptional activity in cells overexpressing SUMO-1 and SUMO-2/3, whereas pretreatment with TAK-981 increased HIF-1α transcriptional activity in response to DMOG. In addition, HIF-1α nuclear accumulation was decreased in cells overexpressing SUMO-1. Importantly, we showed that HIF-1α is not directly SUMOylated, but that SUMOylation affects HIF-1α stability and activity indirectly. Taken together, our results indicate that SUMOylation indirectly suppresses HIF-1α protein stability, transcriptional activity, and nuclear accumulation in intestinal epithelial cells.

Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression.

The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.

Intracellular energy production and distribution in hypoxia.

The hydrolysis of ATP is the primary source of metabolic energy for eukaryotic cells. Under physiological conditions, cells generally produce more than sufficient levels of ATP to fuel the active biological processes necessary to maintain homeostasis. However, mechanisms underpinning the distribution of ATP to subcellular microenvironments with high local demand remain poorly understood. Intracellular distribution of ATP in normal physiological conditions has been proposed to rely on passive diffusion across concentration gradients generated by ATP producing systems such as the mitochondria and the glycolytic pathway. However, subcellular microenvironments can develop with ATP deficiency due to increases in local ATP consumption. Alternatively, ATP production can be reduced during bioenergetic stress during hypoxia. Mammalian cells therefore need to have the capacity to alter their metabolism and energy distribution strategies to compensate for local ATP deficits while also controlling ATP production. It is highly likely that satisfying the bioenergetic requirements of the cell involves the regulated distribution of ATP producing systems to areas of high ATP demand within the cell. Recently, the distribution (both spatially and temporally) of ATP-producing systems has become an area of intense investigation. Here, we review what is known (and unknown) about intracellular energy production and distribution and explore potential mechanisms through which this targeted distribution can be altered in hypoxia, with the aim of stimulating investigation in this important, yet poorly understood field of research.

The HIF-prolyl hydroxylases have distinct and nonredundant roles in colitis-associated cancer.

Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD). HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) control cellular adaptation to hypoxia and are considered promising therapeutic targets in IBD. However, their relevance in the pathogenesis of CAC remains elusive. We induced CAC in Phd1-/-, Phd2+/-, Phd3-/-, and WT mice with azoxymethane (AOM) and dextran sodium sulfate (DSS). Phd1-/- mice were protected against chronic colitis and displayed diminished CAC growth compared with WT mice. In Phd3-/- mice, colitis activity and CAC growth remained unaltered. In Phd2+/- mice, colitis activity was unaffected, but CAC growth was aggravated. Mechanistically, Phd2 deficiency (i) increased the number of tumor-associated macrophages in AOM/DSS-induced tumors, (ii) promoted the expression of EGFR ligand epiregulin in macrophages, and (iii) augmented the signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 signaling, which at least in part contributed to aggravated tumor cell proliferation in colitis-associated tumors. Consistently, Phd2 deficiency in hematopoietic (Vav:Cre-Phd2fl/fl) but not in intestinal epithelial cells (Villin:Cre-Phd2fl/fl) increased CAC growth. In conclusion, the 3 different PHD isoenzymes have distinct and nonredundant effects, promoting (PHD1), diminishing (PHD2), or neutral (PHD3), on CAC growth.

HIF1α-Dependent Induction of TFRC by a Combination of Intestinal Inflammation and Systemic Iron Deficiency in Inflammatory Bowel Disease.

Background and Aims: Iron deficiency (ID) is a frequent extra-intestinal manifestation in patients with Inflammatory Bowel Disease (IBD), who often do not respond to iron supplementation. Iron is a cofactor for hydroxylases that suppress the hypoxia-inducible factor-1α (HIF1α), a transcription factor regulating iron homeostasis. We hypothesized that iron deficiency affects mucosal HIF1α activity in IBD. Methods: IBD patients (n = 101) were subdivided based on iron status (ferritin levels or transferrin saturation) and systemic inflammation (C-reactive protein levels). 154 corresponding ileal and colonic biopsies were analyzed for differential expression of 20 HIF1α pathway-associated genes and related to iron and inflammation status. In vitro expression of selected HIF1α pathway genes were analyzed in wild-type and HIF1A-null Caco-2 cells. Results: Gene expression of the mucosal HIF1α pathway was most affected by intestinal location and inflammatory status. Especially, ileal mucosal TFRC expression, encoding the transferrin receptor TFR1, was increased in inflamed tissue (p < 0.001), and further enhanced in ID. Accordingly, TFRC expression in inflamed tissue associated negatively with serum iron levels, which was not observed in the non-inflamed mucosa. The HIF1α pathway agonist DMOG increased TFRC expression in Caco-2 cells, which was blunted in HIF1A-null cells. Conclusion: We demonstrate that inflammation and anatomical location primarily determine HIF1α pathway activation and downstream TFRC expression in the intestinal mucosa. IBD patients with ID may benefit from treatment with HIF1α-agonists by 1) increasing TFRC-mediated iron absorption in non-inflamed tissue and 2) decreasing mucosal inflammation, thereby improving their responsiveness to oral iron supplementation.

Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Regulation of the Hypoxia-Inducible Factor (HIF) by Pro-Inflammatory Cytokines.

Hypoxia and inflammation are frequently co-incidental features of the tissue microenvironment in a wide range of inflammatory diseases. While the impact of hypoxia on inflammatory pathways in immune cells has been well characterized, less is known about how inflammatory stimuli such as cytokines impact upon the canonical hypoxia-inducible factor (HIF) pathway, the master regulator of the cellular response to hypoxia. In this review, we discuss what is known about the impact of two major pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), on the regulation of HIF-dependent signaling at sites of inflammation. We report extensive evidence for these cytokines directly impacting upon HIF signaling through the regulation of HIF at transcriptional and post-translational levels. We conclude that multi-level crosstalk between inflammatory and hypoxic signaling pathways plays an important role in shaping the nature and degree of inflammation occurring at hypoxic sites.

Inhibition of HIF-prolyl hydroxylases improves healing of intestinal anastomoses.

Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2 haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immunomodulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favorable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.

A Novel RELA Truncating Mutation in a Familial Behçet's Disease-like Mucocutaneous Ulcerative Condition.

OBJECTIVE: Monogenic Behçet's disease (BD)-like conditions are increasingly recognized and to date have been found to predominantly involve loss-of-function variants in TNFAIP3. This study was undertaken to identify genetic and pathobiologic mechanisms associated with a BD-like mucocutaneous ulcerative syndrome and neuromyelitis optica (NMO) occurring in 3 generations of an Irish family (n = 5 cases and 5 familial controls). METHODS: Whole-exome sequencing was used to identify potential pathogenic variants in affected family members and determine segregation between affected and unaffected individuals. Relative v-rel reticuloendotheliosis viral oncogene homolog A (RELA) expression in peripheral blood mononuclear cells was compared by Western blotting. Human epithelial and RelA-/- mouse fibroblast experimental systems were used to determine the molecular impact of the RELA truncation in response to tumor necrosis factor (TNF). NF-κB signaling, transcriptional activation, apoptosis, and cytokine production were compared between wild-type and truncated RELA in experimental systems and patient samples. RESULTS: A heterozygous cytosine deletion at position c.1459 in RELA was detected in affected family members. This mutation resulted in a frameshift p.His487ThrfsTer7, producing a truncated protein disrupting 2 transactivation domains. The truncated RELA protein lacks a full transactivation domain. The RELA protein variants were expressed at equal levels in peripheral mononuclear cells. RelA-/- mouse embryonic fibroblasts (MEFs) expressing recombinant human RELAp.His487ThrfsTer7 were compared to those expressing wild-type RELA; however, there was no difference in RELA nuclear translocation. In RelA-/- MEFs, expression of RELAp.His487ThrfsTer7 resulted in a 1.98-fold higher ratio of cleaved caspase 3 to caspase 3 induced by TNF compared to wild-type RELA (P = 0.036). CONCLUSION: Our data indicate that RELA loss-of-function mutations cause BD-like autoinflammation and NMO via impaired NF-κB signaling and increased apoptosis.

Hypoxia and Innate Immunity: Keeping Up with the HIFsters.

Recent years have witnessed an emergence of interest in understanding metabolic changes associated with immune responses, termed immunometabolism. As oxygen is central to all aerobic metabolism, hypoxia is now recognized to contribute fundamentally to inflammatory and immune responses. Studies from a number of groups have implicated a prominent role for oxygen metabolism and hypoxia in innate immunity of healthy tissue (physiologic hypoxia) and during active inflammation (inflammatory hypoxia). This inflammatory hypoxia emanates from a combination of recruited inflammatory cells (e.g., neutrophils, eosinophils, and monocytes), high rates of oxidative metabolism, and the activation of multiple oxygen-consuming enzymes during inflammation. These localized shifts toward hypoxia have identified a prominent role for the transcription factor hypoxia-inducible factor (HIF) in the regulation of innate immunity. Such studies have provided new and enlightening insight into our basic understanding of immune mechanisms, and extensions of these findings have identified potential therapeutic targets. In this review, we summarize recent literature around the topic of innate immunity and mucosal hypoxia with a focus on transcriptional responses mediated by HIF.

The Shc protein Rai enhances T-cell survival under hypoxia.

Hypoxia occurs in physiological and pathological conditions. T cells experience hypoxia in pathological and physiological conditions as well as in lymphoid organs. Indeed, hypoxia-inducible factor 1α (HIF-1α) affects T cell survival and functions. Rai, an Shc family protein member, exerts pro-survival effects in hypoxic neuroblastoma cells. Since Rai is also expressed in T cells, we here investigated its role in hypoxic T cells. In this work, hypoxia differently affected cell survival, proapoptotic, and metabolic programs in T cells, depending upon Rai expression. By using Jurkat cells stably expressing Rai and splenocytes from Rai-/- mice, we demonstrated that Rai promotes T cell survival and affects cell metabolism under hypoxia. Upon exposure to hypoxia, Jurkat T cells expressing Rai show (a) higher HIF-1α protein levels; (b) a decreased cell death and increased Akt/extracellular-signal-regulated kinase phosphorylation; (c) a decreased expression of proapoptotic markers, including caspase activities and poly(ADP-ribose) polymerase cleavage; (d) an increased glucose and lactate metabolism; (e) an increased activation of nuclear factor-kB pathway. The opposite effects were observed in hypoxic splenocytes from Rai-/- mice. Thus, Rai plays an important role in hypoxic signaling and may be relevant in the protection of T cells against hypoxia.

Prolyl Hydroxylase Inhibition Mitigates Pouchitis.

BACKGROUND: Pouchitis is the most common long-term complication after restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) or familial adenomatous polyposis (FAP), which can eventually progress to pouch failure, necessitating permanent stoma construction. Hypoxia-inducible transcription factor prolyl hydroxylase-containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors that control adaptive gene expression through hypoxia-inducible factor (HIF). Emerging evidence supports PHDs as being therapeutic targets in intestinal inflammation. However, pharmacological inhibition of PHDs has not been validated as a treatment strategy in pouchitis. METHODS: PHD1-3 mRNA and protein expression were analyzed in mucosal pouch and prepouch ileal patient biopsies. After establishment of a preclinical IPAA model in rats, the impact of the pan-PHD small-molecule inhibitor dimethyloxalylglycine (DMOG) on dextran sulfate sodium (DSS)-induced pouchitis was studied. Clinical and molecular parameters were investigated. RESULTS: PHD1, but not PHD2 or PHD3, was overexpressed in pouchitis in biopsies of patients with IPAA for UC but not FAP. In addition, PHD1 expression correlated with disease activity. DMOG treatment profoundly mitigated DSS-induced pouchitis in a rodent IPAA model. Mechanistically, DMOG restored intestinal epithelial barrier function by induction of tight junction proteins zona occludens-1 and claudin-1 and alleviation of intestinal epithelial cell apoptosis, thus attenuating pouch inflammation. CONCLUSIONS: Together, these results establish a strong therapeutic rationale for targeting PHD1 with small-molecule inhibitors in pouchitis after IPAA for UC.

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extracellular opportunistic bacterial pathogen commonly associated with infectious complications in susceptible individuals, such as those with underlying diseases including HIV/AIDS and cystic fibrosis. Antibiotic resistance in multiple strains of P. aeruginosa is a rapidly developing clinical problem. We have previously demonstrated that the oxygen levels at the site of P. aeruginosa infection can strongly influence virulence and antibiotic resistance in this pathogen, although the oxygen-sensing and -signaling mechanisms underpinning this response have remained unknown. In this study, we investigated the potential role of the putative oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) in the control of virulence and antibiotic resistance in P. aeruginosa We found that a P. aeruginosa strain lacking PPHD (PAO310) exhibits increased virulence associated with increased bacterial motility. Furthermore, PPHD-deficient P. aeruginosa displayed enhanced antibiotic resistance against tetracycline through increased expression of the xenobiotic transporters mexEF-oprN and MexXY. Of note, the effect of the PPHD knockout on antibiotic resistance was phenocopied in bacteria exposed to atmospheric hypoxia. We conclude that PPHD is a putative bacterial oxygen sensor that may link microenvironmental oxygen levels to virulence and antibiotic resistance in P. aeruginosa.

The PHD1 oxygen sensor in health and disease.

The hypoxia-inducible factor (HIF) co-ordinates the adaptive transcriptional response to hypoxia in metazoan cells. The hypoxic sensitivity of HIF is conferred by a family of oxygen-sensing enzymes termed HIF hydroxylases. This family consists of three prolyl hydroxylases (PHD1-3) and a single asparagine hydroxylase termed factor inhibiting HIF (FIH). It has recently become clear that HIF hydroxylases are functionally non-redundant and have discrete but overlapping physiological roles. Furthermore, altered abundance or activity of these enzymes is associated with a number of pathologies. Pharmacological HIF-hydroxylase inhibitors have recently proven to be both tolerated and therapeutically effective in patients. In this review, we focus on the physiology, pathophysiology and therapeutic potential of the PHD1 isoform, which has recently been implicated in diseases including inflammatory bowel disease, ischaemia and cancer.

Regulation of immunity and inflammation by hypoxia in immunological niches.

Immunological niches are focal sites of immune activity that can have varying microenvironmental features. Hypoxia is a feature of physiological and pathological immunological niches. The impact of hypoxia on immunity and inflammation can vary depending on the microenvironment and immune processes occurring in a given niche. In physiological immunological niches, such as the bone marrow, lymphoid tissue, placenta and intestinal mucosa, physiological hypoxia controls innate and adaptive immunity by modulating immune cell proliferation, development and effector function, largely via transcriptional changes driven by hypoxia-inducible factor (HIF). By contrast, in pathological immunological niches, such as tumours and chronically inflamed, infected or ischaemic tissues, pathological hypoxia can drive tissue dysfunction and disease development through immune cell dysregulation. Here, we differentiate between the effects of physiological and pathological hypoxia on immune cells and the consequences for immunity and inflammation in different immunological niches. Furthermore, we discuss the possibility of targeting hypoxia-sensitive pathways in immune cells for the treatment of inflammatory disease.

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

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.