Inflammation and Hypoxia: HIF and PHD Isoform Selectivity
Authors: Emily R. Watts, Sarah R. Walmsley
Journal: Trends in Molecular Medicine (2018)

Hypoxia signaling pathways are highly conserved between species and are ubiquitously expressed in mammalian cells. Activation of HIF has pleiotropic effects, and is under investigation as a potential therapeutic target in malignancy, ischemia, and inflammatory disease processes. Hypoxia and inflammation frequently coexist and may exacerbate one another. There are multiple and bidirectional links between the molecular pathways which sense and respond to hypoxia and inflammatory signals. Activation or inhibition of the HIF–PHD pathway is subject to fine-tuning through isoform-specific functions and expression profiles for different HIFs and PHDs. Early evidence from mouse studies supports the view that targeting this pathway may be therapeutic in inflammation, although the risks of off-target or side effects must be considered. Cautious optimism is urged. Cells sense and respond to hypoxia through the activity of the transcription factor HIF (hypoxia-inducible factor) and its regulatory hydroxylases, the prolyl hydroxylase domain enzymes (PHDs). Multiple isoforms of HIFs and PHDs exist, and isoform-selective roles have been identified in the context of the inflammatory environment, which is itself frequently hypoxic. Recent advances in the field have highlighted the complexity of this system, particularly with regards to the cell and context-specific activity of HIFs and PHDs. Because novel therapeutic agents which regulate this pathway are nearing the clinic, understanding the role of HIFs and PHDs in inflammation outcomes is an essential step in avoiding off-target effects and, crucially, in developing new anti-inflammatory strategies. Cells sense and respond to hypoxia through the activity of the transcription factor HIF (hypoxia-inducible factor) and its regulatory hydroxylases, the prolyl hydroxylase domain enzymes (PHDs). Multiple isoforms of HIFs and PHDs exist, and isoform-selective roles have been identified in the context of the inflammatory environment, which is itself frequently hypoxic. Recent advances in the field have highlighted the complexity of this system, particularly with regards to the cell and context-specific activity of HIFs and PHDs. Because novel therapeutic agents which regulate this pathway are nearing the clinic, understanding the role of HIFs and PHDs in inflammation outcomes is an essential step in avoiding off-target effects and, crucially, in developing new anti-inflammatory strategies. Cellular responses to hypoxia are essential for life and are now recognized to be dysregulated in a wide range of disease processes. The key players in these pathways are a family of transcription factors, the hypoxia-inducible factors (HIFs; see Glossary), as well as their regulatory proteins, the prolyl hydroxylase domain enzymes (PHDs) and factor inhibiting HIF (FIH, also known as HIF1α subunit inhibitor, HIF1AN) (Figure 1). The PHDs are also termed EGLNs owing to their early identification as homologs of the C. elegans oxygen sensor EGL-nine. In this nomenclature PHD1 is termed EGLN2, PHD2 is termed EGLN1, and PHD3 is known as EGLN3. There are multiple links between hypoxia signaling pathways and inflammatory processes. Inflammation may lead to local or systemic hypoxia, for example in an abscess where lack of blood supply results in profound local hypoxia, or in acute lung inflammation (such as acute respiratory distress syndrome, ARDS) where impaired gas exchange can lead to systemic hypoxia. In addition, an influx of inflammatory cells may lead to hypoxia because the demand for oxygen outstrips supply, so-called inflammatory hypoxia [1Campbell E.L. et al.Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.Immunity. 2014; 40: 66-77Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar]. The links between hypoxia signaling and inflammation are bidirectional: hypoxia may also exacerbate inflammation through activation of inflammatory pathways and effects on immune cell fate and function. The HIF/PHD pathway can also be activated in normoxia in response to inflammatory stimuli. For example, lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, has been shown to upregulate HIF1α in macrophages [2Blouin C.C. et al.Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1alpha.Blood. 2004; 103: 1124-1130Crossref PubMed Scopus (363) Google Scholar], and T cell receptor (TCR) engagement contributes to HIF1α and HIF2α (also known as endothelial PAS domain protein 1, EPAS1) protein accumulation in T cells [3Nakamura H. et al.TCR engagement increases hypoxia-inducible factor-1 alpha protein synthesis via rapamycin-sensitive pathway under hypoxic conditions in human peripheral T cells.J. Immunol. 2005; 174: 7592-7599Crossref PubMed Scopus (117) Google Scholar, 4Doedens A.L. et al.Hypoxia-inducible factors enhance the effector responses of CD8+ T cells to persistent antigen.Nat Immunol. 2013; 14: 1173-1182Crossref PubMed Scopus (407) Google Scholar]. It is also important to consider that the PHDs have been postulated to have HIF-independent activity in the context of inflammation and malignancy. NF-κB is a transcription factor which, like HIF, regulates a diverse group of genes including cytokines, immune receptors, and stress-response genes [5Pahl H.L. Activators and target genes of Rel/NF-kappaB transcription factors.Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3446) Google Scholar]. In some contexts NF-κB activity is linked to hypoxia signaling in a HIF-dependent fashion [6Rius J. et al.NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha.Nature. 2008; 453: 807-811Crossref PubMed Scopus (1163) Google Scholar, 7Walmsley S.R. et al.Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity.J. Exp. Med. 2005; 201: 105-115Crossref PubMed Scopus (660) Google Scholar]. 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FIH has also been found to regulate NF-κB activity, and Fih (Hif1an) and Phd1 (Egln2) knockouts had an additive effect in reducing NF-κB activity [11Scholz C.C. et al.Regulation of IL-1β-induced NF-κB by hydroxylases links key hypoxic and inflammatory signaling pathways.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 18490-18495Crossref PubMed Scopus (121) Google Scholar]. Furthermore, PHD activity has been found to have HIF-independent effects on cell proliferation and survival via PHD1-dependent hydroxylation of the tumor-suppressor protein p53 [12Deschoemaeker S. et al.PHD1 regulates p53-mediated colorectal cancer chemoresistance.EMBO Mol. 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Despite similarities in structure and function of HIFα isoforms, there are key differences in the transcriptional repertoires of HIF1α and HIF2α which afford an additional level of complexity in the transcriptional response to hypoxia (reviewed in [15Dengler V.L. et al.Transcriptional regulation by hypoxia inducible factors.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 1-15Crossref PubMed Scopus (420) Google Scholar]). This differentiation is multifactorial, relating to variations in tissue expression, temporal regulation, and gene specificity. Interestingly, this specificity is driven not by the DNA-binding domain but by the N-terminal transactivation domain [16Hu C.-J. et al.The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha.Mol. Biol. Cell. 2007; 18: 4528-4542Crossref PubMed Scopus (272) Google Scholar]. The balance of HIF1α versus HIF2α has important implications for inflammation. PHDs 1–3 were identified in 2001 as the oxygen sensors regulating HIF [17Epstein A.C. et al.C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2729) Google Scholar, 18Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2112) Google Scholar]. All three mammalian PHD isoforms have HIF hydroxylation activity [17Epstein A.C. et al.C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2729) Google Scholar]. The specific function of each HIF and PHD isoform is the subject of extensive and ongoing research. The generation of cell- and isoform-specific knockout mice (often using Cre–lox recombination [19Sauer B. Inducible gene targeting in mice using the Cre/lox system.Methods. 1998; 14: 381-392Crossref PubMed Scopus (595) Google Scholar]) and other techniques such as small interfering RNAs (siRNAs) [20Carthew R.W. Sontheimer E.J. Origins and mechanisms of miRNAs and siRNAs.Cell. 2009; 136: 642-655Abstract Full Text Full Text PDF PubMed Scopus (3804) Google Scholar] have enabled researchers to begin to elucidate the function of specific PHDs. These studies, as well as those utilizing pharmacological inhibition of this pathway, identify diverse and complex roles for individual pathway members in the inflammatory response. Hypoxia signaling pathways are active in almost all mammalian cells, including immune cells and tissues such as the epithelium and endothelium. This is of crucial importance when considering the influence of environmental hypoxia in inflammatory conditions and is particularly relevant in the specialized tissues of the intestine, lung, and joint, all of which may be the site of infection, sterile inflammation (owing to physical, chemical, or metabolic stressors [21Rubartelli A. et al.Mechanisms of sterile inflammation.Front. Immunol. 2013; 4: 398Crossref PubMed Scopus (38) Google Scholar]), or autoimmune disease, and are also subject to local hypoxia. This hypoxia may be due to pathology but can also occur in normal physiology, as in the intestine. HIF1α, HIF2α, PHD1, and PHD3 have all been implicated in regulation of intestinal inflammation [22Sun M. et al.Hypoxia inducible factor-1α-induced interleukin-33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease.Clin. Exp. 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Use of Phd1 knockout models targeting myeloid and/or endothelial cells also identified a specific role for hematopoietic PHD1 deficiency in protecting against colitis [27Van Welden S. et al.Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.J. Pathol. 2017; 241: 547-558Crossref PubMed Scopus (23) Google Scholar]. In contrast to these findings, Phd3 deletion in intestinal epithelial cells (IECs) was associated with increased susceptibility to colitis [26Chen Y. et al.PHD3 stabilizes the tight junction protein occludin and protects intestinal epithelial barrier function.J. Biol. Chem. 2015; 290: 20580-20589Crossref PubMed Scopus (43) Google Scholar]. Knockout mice developed both spontaneous colitis and more severe DSS-induced disease than controls. PHD3 deficiency does not alter IEC apoptosis, but is required for the synthesis of occludin protein which maintains IEC tight-junction integrity. Furthermore, in biopsies from patients with ulcerative colitis, PHD3 levels were inversely correlated with disease severity. HIF1α and HIF2α also appear to have differential functions in the intestinal epithelium. Broadly speaking, IEC HIF1α is found to be protective via induction of IL-33 and upregulation of pro-resolution factors [22Sun M. et al.Hypoxia inducible factor-1α-induced interleukin-33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease.Clin. Exp. Immunol. 2017; 187: 428-440Crossref PubMed Scopus (27) Google Scholar]. In addition, hypoxia itself is protective through HIF1α-mediated induction of autophagy in IECs and down regulation of inflammatory cytokines [28Cosin-Roger J. et al.Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation.Nat Commun. 2017; 8: 98Crossref PubMed Scopus (183) Google Scholar]. On the other hand, IEC-specific Hif2a knockout mice were protected from DSS-induced colitis, whereas HIF2α overexpression led to spontaneous colitis [23Xue X. et al.Endothelial PAS domain protein 1 activates the inflammatory response in the intestinal epithelium to promote colitis in mice.Gastroenterology. 2013; 145: 831-841Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. In addition, epithelial HIF2α was shown to promote colon carcinogenesis via its capacity to enhance neutrophil recruitment [29Triner D. et al.Epithelial hypoxia-inducible factor 2α facilitates the progression of colon tumors through recruiting neutrophils.Mol. Cell. Biol. 2017; 37 (e00481-16)Crossref PubMed Scopus (40) Google Scholar]. Pan-hydroxylase inhibition has been posited as a novel therapy in IBD (reviewed in [30Eltzschig H.K. et al.Targeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases.Nat. Rev. Drug Discov. 2014; 13: 852-869Crossref PubMed Scopus (247) Google Scholar]), and encouraging results have been obtained in mouse models [31Marks E. et al.Oral delivery of prolyl hydroxylase inhibitor: AKB-4924 promotes localized mucosal healing in a mouse model of colitis.Inflamm. Bowel Dis. 2015; 21: 267-275Crossref PubMed Scopus (39) Google Scholar, 32Keely S. et al.Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis.Mucosal Immunol. 2014; 7: 114-123Crossref PubMed Scopus (87) Google Scholar, 33Robinson A. et al.Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition.Gastroenterology. 2008; 134: 145-155Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar]. However, the divergent effects of PHD1, PHD3, and of the different HIFα isoforms require careful consideration: strategies that inhibit PHD1 or enhance HIF1α activity would be predicted to be beneficial, but if these same strategies were to limit PHD3 activity or increase HIF2α stabilization the effects may be detrimental. Understanding which PHD or HIF isoform is the dominant form in a particular context is essential. Differential isoform functions are also seen in inflammatory joint conditions: immunostaining of synovial biopsies from patients with rheumatoid arthritis (RA) showed that HIF2α, but not HIF1α, was highly expressed in this setting: >60% of the area stained was positive for HIF2α, while <10% was positive for HIF1α. Furthermore, HIF2α colocalized with inflammatory markers in both RA biopsies and in murine RA models [34Ryu J.-H. et al.Hypoxia-inducible factor-2α is an essential catabolic regulator of inflammatory rheumatoid arthritis.PLoS Biol. 2014; 12e1001881Crossref PubMed Scopus (67) Google Scholar]. HIF2α expression was specific for RA because biopsies from patients with osteoarthritis showed <10% staining for both HIF isoforms. In mice, HIF2α overexpression led to arthritis while HIF2α deficiency was protective. Increased HIF2α in the synovium led to increased IL-6 production and polarization of inflammatory T helper 17 (TH17) cells, suggesting that environmental, as well as immune cell-intrinsic, HIF/PHDs can polarize the immune response (discussed further below). A study of PHD isoforms in inflamed joints identified PHD2 as the predominant regulator of HIF1α and HIF2α in this context [35Muz B. et al.Prolyl hydroxylase domain enzyme 2 is the major player in regulating hypoxic responses in rheumatoid arthritis.Arthritis Rheum. 2012; 64: 2856-2867Crossref PubMed Scopus (30) Google Scholar], and upregulation of inflammatory and angiogenic mediators was found in response to siRNA knockdown of PHD2. However, the inflamed synovium does not exist in isolation, and infiltrating immune cells of both the innate and adaptive lineages are essential in the development of joint inflammation (reviewed in [36Nevius E. et al.Inflammatory cell migration in rheumatoid arthritis: a comprehensive review.Clin. Rev. Allergy Immunol. 2016; 51: 59-78Crossref PubMed Scopus (59) Google Scholar]). Thus, as in the intestine, the overall effect of hypoxia (and indeed of targeting hypoxia-sensitive pathways) on synovial and epithelial cells must be weighed against the impact of hypoxia on the migrating immune cells. The lung represents a unique organ in terms of oxygen and pathogen exposure. Whereas in health the lung is exposed to high oxygen levels from inspired air, in pathology areas of marked local hypoxia can still occur. Like the intestine, the lung is exposed to an enormous range of foreign but innocuous material, and therefore must have robust immune-tolerance mechanisms. However, it is also susceptible to both sterile inflammation and infection with pathogens including viruses, bacteria, and fungi. HIF1α is stabilized in alveolar epithelial cells in response to inflammatory stimuli [37McClendon J. et al.Hypoxia-inducible factor 1α signaling promotes repair of the alveolar epithelium after acute lung injury.Am. J. Pathol. 2017; 187: 1772-1786Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. Resolution and repair in the alveolus is dependent upon the proliferation and transdifferentiation of alveolar cells, and HIF1α-dependent VEGF expression was found to be crucial for this response [37McClendon J. et al.Hypoxia-inducible factor 1α signaling promotes repair of the alveolar epithelium after acute lung injury.Am. J. Pathol. 2017; 187: 1772-1786Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. HIF1α was also stabilized in response to stretch injury, consistent with mechanical ventilation. Interestingly, this normoxic induction of HIF was also found to be protective in acute lung injury (ALI) models [38Eckle T. et al.HIF1A reduces acute lung injury by optimizing carbohydrate metabolism in the alveolar epithelium.PLoS Biol. 2013; 11e1001665Crossref PubMed Scopus (114) Google Scholar]. HIFα stabilization was dependent on induction of succinate dehydrogenase and consequent inhibition of PHD activity. Alveolar epithelial cell-specific Hif1a deletion is associated with increased morbidity and mortality in stretch-induced ALI. In contrast to these findings, Xi et al. [39Xi Y. et al.Local lung hypoxia determines epithelial fate decisions during alveolar regeneration.Nat. Cell Biol. 2017; 19: 904-914Crossref PubMed Scopus (142) Google Scholar] found that, in the context of influenza-induced alveolar epithelial cell loss, HIF1α activation may be associated with dysfunctional alveolar remodeling. The authors found that hypoxia, dependent on HIF1α activity, was key in determining epithelial cell fate, and that mice lacking epithelial cell HIF1α recovered more quickly, with improved expansion of the type II alveolar cell population and restoration of normal epithelium. In addition to these findings focusing on acute lung injury, both HIF1α and HIF2α in the lung epithelium have been shown to play a role in chronic, cobalt-induced, lung inflammation [40Saini Y. et al.Acute cobalt-induced lung injury and the role of hypoxia-inducible factor 1alpha in modulating inflammation.Toxicol. Sci. 2010; 116: 673-681Crossref PubMed Scopus (28) Google Scholar, 41Proper S.P. et al.Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.Toxicol. Sci. 2014; 137: 447-457Crossref PubMed Scopus (14) Google Scholar]. Loss of HIF1α was associated with a switch from type 1 inflammation (characterized by cytokines such as TNF-α and IL-2) to type 2 (allergic-type) inflammation with fewer neutrophils but increased eosinophilic inflammation and increased TH2 cytokines such as IL-4 [40Saini Y. et al.Acute cobalt-induced lung injury and the role of hypoxia-inducible factor 1alpha in modulating inflammation.Toxicol. Sci. 2010; 116: 673-681Crossref PubMed Scopus (28) Google Scholar]. Hfi2a deletion was similarly associated with enhanced and prolonged eosinophilic infiltration [41Proper S.P. et al.Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.Toxicol. Sci. 2014; 137: 447-457Crossref PubMed Scopus (14) Google Scholar]. Interestingly, the time-frame of this eosinophilic inflammation varied depending on which HIFα isoform was targeted, consistent with other studies showing that the predominant isoform varies over the timecourse of an inflammatory response [42Thompson A.A.R. et al.Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish.Blood. 2014; 123: 366-376Crossref PubMed Scopus (95) Google Scholar], with HIF2α appearing to predominate in the latter, reparative stages of inflammation in the lung. These data together suggest that potentiating the effects of HIF1α in intestinal or lung epithelial cells may be an appealing therapeutic strategy, but caution must be exercised because any benefit must be weighed against potential harm caused by altering hypoxia signaling in immune cells, which we discuss in detail below. Infiltrating immune cells are a key component of any inflammatory response, and their behavior shapes the phenotype, severity, and duration of the response. Hypoxia and HIF/PHD activity have been shown to have profound effects on cells of both the innate and adaptive immune systems. Both macrophages and neutrophils have been shown to express HIF1α and HIF2α in response to hypoxia [42Thompson A.A.R. et al.Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish.Blood. 2014; 123: 366-376Crossref PubMed Scopus (95) Google Scholar, 43Fang H.-Y. et al.Hypoxia-inducible factors 1 and 2 are important transcriptional effectors in primary macrophages experiencing hypoxia.Blood. 2009; 114: 844-859Crossref PubMed Scopus (230) Google Scholar]. Hypoxic survival is a key neutrophil response which enables these immune 'first responders' to function in hostile environments; however, inappropriate HIF activation leading to neutrophil persistence may contribute to delayed inflammation resolution and tissue damage. HIF1α has been shown to be essential for neutrophil survival in hypoxia [7Walmsley S.R. et al.Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity.J. Exp. Med. 2005; 201: 105-115Crossref PubMed Scopus (660) Google Scholar], while HIF2α promotes neutrophil persistence during sterile inflammation, indicative of context-specific roles for these isoforms. Furthermore, distinct temporal expression profiles [42Thompson A.A.R. et al.Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish.Blood. 2014; 123: 366-376Crossref PubMed Scopus (95) Google Scholar], with early HIF1α upregulation and delayed HIF2α induction suggest functional divergence during different phases of the inflammatory response. Given this crucial role for HIF in neutrophil survival and function, it is of little surprise that manipulation of PHD activity has important consequences for neutrophil survival and function. Myeloid-specific genetic deletion of Phd2 or pharmacological inhibition of PHD2 activity resulted in exaggerated neutrophilic inflammation in response to Streptococcus pneumoniae and in an LPS-induced acute lung injury model. This was not due to failure of pathogen clearance but instead to an over-exuberant neutrophil response, with increased migration, enhanced survival, and upregulation of glycolysis with consequent increased energy charge [44Sadiku P. et al.Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.J. Clin. Invest. 2017; 127: 3407-3420Crossref PubMed Scopus (57) Google Scholar]. Importantly, these findings were associated with increased stability of HIF1α. In contrast to these findings, loss of PHD3 in neutrophils was associated with reduced inflammation [45Walmsley S.R. et al.Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice.J. Clin. Invest. 2011; 121: 1053-1063Crossref PubMed Scopus (132) Google Scholar]. Both hypoxia and inflammatory stimuli were found to increase levels of PHD3 in neutrophils (consistent with findings in other cell types). Interestingly, despite preserved HIF transcriptional activity, loss of PHD3 led to a failure of neutrophil hypoxic survival. This had important consequences in vivo, with improved resolution of LPS-induced acute lung injury and reduced inflammation in a colitis model. Seminal work by Cramer et al. demonstrated the essential role of HIF1α in myeloid cells [46Cramer T. et al.HIF-1alpha is essential for myeloid cell-mediated inflammation.Cell. 2003; 112: 645-657Abstract Full Text Full Text PDF PubMed Scopus (1604) Google Scholar]. More recent studies have begun to unpick the underlying mechanisms, with particular emphasis on the link between hypoxia signaling pathways, metabolism, and polarization states. Different HIFα isoforms may regulate the polarization state of macrophages (and vice versa), with consequences for inflammation outcome (Figure 2A). Levels of HIF1α and HIF2α are differentially regulated by TH1/M1 and TH2/M2 cytokines, and M1 signals such as IFN-γ and LPS promote HIF1α expression, whereas IL-4 and IL-13 (M2-promoting signals) favor HIF2α accumulation [47Takeda N. et al.Differential activation and antagonistic function of HIF-{alpha} isoforms in macrophages are essential for NO homeostasis.Genes Dev. 2010; 24: 491-501Crossref PubMed Scopus (445) Google Scholar]. In keeping with the notion that HIF1α drives an inflammatory phenotype, whereas HIF2α is more associated with an M2 phenotype, Dehn et al. [48Dehn S. et al.HIF-2α in resting macrophages tempers mitochondrial reactive oxygen species to selectively repress MARCO-dependent phagocytosis.J. Immunol. 2016; 197: 3639-3649Crossref PubMed Scopus (18) Google Scholar] demonstrated that, although HIF1α was required for macrophage phagocytosis under hypoxic conditions, HIF2α was not essential. HIF1α was crucial for iNOS generation, a key marker of M1 macrophages. Conversely, HIF2α regulated the M2 gene arginase 1 (Arg1). Not only was HIF1α stabilized by M1 signals, but its activity was also e