Insulin signaling establishes a developmental trajectory of adipose regulatory T cells.

Systematic characterizations of adipose regulatory T (Treg) cell subsets and their phenotypes remain uncommon. Using single-cell ATAC-sequencing and paired single-cell RNA and T cell receptor (TCR) sequencing to map mouse adipose Treg cells, we identified CD73hiST2lo and CD73loST2hi subsets with distinct clonal expansion patterns. Analysis of TCR-sharing data implied a state transition between CD73hiST2lo and CD73loST2hi subsets. Mechanistically, we revealed that insulin signaling occurs through a HIF-1α-Med23-PPAR-γ axis to drive the transition of CD73hiST2lo into a CD73loST2hi adipose Treg cell subset. Treg cells deficient in insulin receptor, HIF-1α or Med23 have decreased PPAR-γ expression that in turn promotes accumulation of CD73hiST2lo adipose Treg cells and physiological adenosine production to activate beige fat biogenesis. We therefore unveiled a developmental trajectory of adipose Treg cells and its dependence on insulin signaling. Our findings have implications for understanding the dynamics of adipose Treg cell subsets in aged and obese contexts.

Skin deep: Epithelial cell metabolism and chronic skin inflammation.

Skin inflammation is potentiated by coordinated epithelial and immune cell metabolism. In this issue of Immunity, Subudhi and Konieczny et al. delineate how HIF1α regulates epithelial cell glycolysis during psoriasis. In turn, lactate is a byproduct that augments type 17 γδ T cell responses to sustain inflammatory skin disease.

Metabolic coordination between skin epithelium and type 17 immunity sustains chronic skin inflammation.

Inflammatory epithelial diseases are spurred by the concomitant dysregulation of immune and epithelial cells. How these two dysregulated cellular compartments simultaneously sustain their heightened metabolic demands is unclear. Single-cell and spatial transcriptomics (ST), along with immunofluorescence, revealed that hypoxia-inducible factor 1α (HIF1α), downstream of IL-17 signaling, drove psoriatic epithelial remodeling. Blocking HIF1α in human psoriatic lesions ex vivo impaired glycolysis and phenocopied anti-IL-17 therapy. In a murine model of skin inflammation, epidermal-specific loss of HIF1α or its target gene, glucose transporter 1, ameliorated epidermal, immune, vascular, and neuronal pathology. Mechanistically, glycolysis autonomously fueled epithelial pathology and enhanced lactate production, which augmented the γδ T17 cell response. RORγt-driven genetic deletion or pharmacological inhibition of either lactate-producing enzymes or lactate transporters attenuated epithelial pathology and IL-17A expression in vivo. Our findings identify a metabolic hierarchy between epithelial and immune compartments and the consequent coordination of metabolic processes that sustain inflammatory disease.

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

Into Thin Air: How We Sense and Respond to Hypoxia.

This year's Lasker Basic Medical Research Award is shared by William Kaelin, Peter Ratcliffe, and Gregg Semenza for discovery of the pathway by which animal cells sense and adapt to changes in oxygen availability, which plays an essential role in adaptation to a wide variety of physiologic and pathologic conditions.

Paracrine Induction of HIF by Glutamate in Breast Cancer: EglN1 Senses Cysteine.

The HIF transcription factor promotes adaptation to hypoxia and stimulates the growth of certain cancers, including triple-negative breast cancer (TNBC). The HIFα subunit is usually prolyl-hydroxylated by EglN family members under normoxic conditions, causing its rapid degradation. We confirmed that TNBC cells secrete glutamate, which we found is both necessary and sufficient for the paracrine induction of HIF1α in such cells under normoxic conditions. Glutamate inhibits the xCT glutamate-cystine antiporter, leading to intracellular cysteine depletion. EglN1, the main HIFα prolyl-hydroxylase, undergoes oxidative self-inactivation in the absence of cysteine both in biochemical assays and in cells, resulting in HIF1α accumulation. Therefore, EglN1 senses both oxygen and cysteine.

Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages.

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection.

Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/ß-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of ß-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, ß-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted ß-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This ß-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by ß-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a-/- NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.

Oligodendrocyte-encoded HIF function couples postnatal myelination and white matter angiogenesis.

Myelin sheaths provide critical functional and trophic support for axons in white matter tracts of the brain. Oligodendrocyte precursor cells (OPCs) have extraordinary metabolic requirements during development as they differentiate to produce multiple myelin segments, implying that they must first secure adequate access to blood supply. However, mechanisms that coordinate myelination and angiogenesis are unclear. Here, we show that oxygen tension, mediated by OPC-encoded hypoxia-inducible factor (HIF) function, is an essential regulator of postnatal myelination. Constitutive HIF1/2α stabilization resulted in OPC maturation arrest through autocrine activation of canonical Wnt7a/7b. Surprisingly, such OPCs also show paracrine activity that induces excessive postnatal white matter angiogenesis in vivo and directly stimulates endothelial cell proliferation in vitro. Conversely, OPC-specific HIF1/2α loss of function leads to insufficient angiogenesis in corpus callosum and catastrophic axon loss. These findings indicate that OPC-intrinsic HIF signaling couples postnatal white matter angiogenesis, axon integrity, and the onset of myelination in mammalian forebrain.

Increased adipocyte O2 consumption triggers HIF-1α, causing inflammation and insulin resistance in obesity.

Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. Here, we report that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1α induction, setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1α can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance.

Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.

TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains.

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia.

The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.

Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging.

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/ß-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/ß-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor.

Macrophages form a major cell population in the tumor microenvironment. They can be activated and polarized into tumor-associated macrophages (TAM) by the tumor-derived soluble molecules to promote tumor progression and metastasis. Here, we used comparative metabolomics coupled with biochemical and animal studies to show that cancer cells release succinate into their microenvironment and activate succinate receptor (SUCNR1) signaling to polarize macrophages into TAM. Furthermore, the results from in vitro and in vivo studies revealed that succinate promotes not only cancer cell migration and invasion but also cancer metastasis. These effects are mediated by SUCNR1-triggered PI3K-hypoxia-inducible factor 1α (HIF-1α) axis. Compared with healthy subjects and tumor-free lung tissues, serum succinate levels and lung cancer SUCNR1 expression were elevated in lung cancer patients, suggesting an important clinical relevance. Collectively, our findings indicate that the secreted tumor-derived succinate belongs to a novel class of cancer progression factors, controlling TAM polarization and promoting tumorigenic signaling.

HIF: a master regulator of nutrient availability and metabolic cross-talk in the tumor microenvironment.

A role for hypoxia-inducible factors (HIFs) in hypoxia-dependent regulation of tumor cell metabolism has been thoroughly investigated and covered in reviews. However, there is limited information available regarding HIF-dependent regulation of nutrient fates in tumor and stromal cells. Tumor and stromal cells may generate nutrients necessary for function (metabolic symbiosis) or deplete nutrients resulting in possible competition between tumor cells and immune cells, a result of altered nutrient fates. HIF and nutrients in the tumor microenvironment (TME) affect stromal and immune cell metabolism in addition to intrinsic tumor cell metabolism. HIF-dependent metabolic regulation will inevitably result in the accumulation or depletion of essential metabolites in the TME. In response, various cell types in the TME will respond to these hypoxia-dependent alterations by activating HIF-dependent transcription to alter nutrient import, export, and utilization. In recent years, the concept of metabolic competition has been proposed for critical substrates, including glucose, lactate, glutamine, arginine, and tryptophan. In this review, we discuss how HIF-mediated mechanisms control nutrient sensing and availability in the TME, the competition for nutrients, and the metabolic cross-talk between tumor and stromal cells.