Asis Palazon

HIF transcription factors, inflammation, and immunity.

The hypoxic response in cells and tissues is mediated by the family of hypoxia-inducible factor (HIF) transcription factors; these play an integral role in the metabolic changes that drive cellular adaptation to low oxygen availability. HIF expression and stabilization in immune cells can be triggered by hypoxia, but also by other factors associated with pathological stress: e.g., inflammation, infectious microorganisms, and cancer. HIF induces a number of aspects of host immune function, from boosting phagocyte microbicidal capacity to driving T cell differentiation and cytotoxic activity. Cellular metabolism is emerging as a key regulator of immunity, and it constitutes another layer of fine-tuned immune control by HIF that can dictate myeloid cell and lymphocyte development, fate, and function. Here we discuss how oxygen sensing in the immune microenvironment shapes immunological response and examine how HIF and the hypoxia pathway control innate and adaptive immunity.

S -2-hydroxyglutarate regulates CD8 + T-lymphocyte fate

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic–epigenetic axis, to immune fate and function.

Constitutive Glycolytic Metabolism Supports CD8(+) T Cell Effector Memory Differentiation during Viral Infection.

Extensive metabolic changes accompany Txa0cell activation, including a switch to glycolytic energy production and increased biosynthesis. Recent studies suggest that subsequent return to reliance on oxidative phosphorylation and increasing spare respiratory capacity are essential for the differentiation of memory CD8+ Txa0cells. In contrast, we found that constitutive glycolytic metabolism and suppression of oxidative phosphorylation in CD8+ Txa0cells, achieved by conditional deletion of hypoxia-inducible factor regulator Vhl, accelerated CD8+ memory cell differentiation during viral infection. Despite sustained glycolysis, CD8+ memory cells emerged that upregulated key memory-associated cytokine receptors and transcription factors and showed a heightened response to secondary challenge. In addition, increased glycolysis not only permitted memory formation, but it also favored the formation of long-lived effector-memory CD8+ Txa0cells. These data redefine the role of cellular metabolism in memory cell differentiation, showing that reliance on glycolytic metabolism does not hinder formation of a protective memory population.

Immune Response Regulation in the Tumor Microenvironment by Hypoxia

Lymphocytes and myeloid cells sense hypoxia by the hypoxia-inducible factor (HIF) transcriptional system and via other molecular mechanisms. Low O2 availability is a hallmark of most solid tumors in which infiltrating leukocytes experience severe hypoxia once away from nurturing blood vessels. HIF controls migration, differentiation, and effector functions on immune cells. Importantly, in the tumor microenvironment the hypoxia response modulates the expression levels for important molecular targets in immunotherapy such as CD137, OX-40, FOXP3, and PD-L1. Modulation by hypoxia of tumor-associated macrophages, myeloid-derived suppressor cells, and dendritic cells ought to play an important underexplored role in modulating tumor immunity. Overall, low O2 seems to invigorate some anti-tumor effector T-cell functions while conflictingly favoring T-regulatory cells (Tregs) in terms of their differentiation, suppressive functions, and recruitment. Hypoxia also has been shown to uphold myeloid cell-mediated tumor-promoting inflammation and the immunosuppressive functions of tumor-associated macrophages. Detailed research of this intricate and poorly understood balance is warranted to improve the outcome of cancer immunotherapy.

An HIF-1α/VEGF-A Axis in Cytotoxic T Cells Regulates Tumor Progression

Summary Cytotoxic T cells infiltrating tumors are thought to utilize HIF transcription factors during adaptation to the hypoxic tumor microenvironment. Deletion analyses of the two key HIF isoforms found that HIF-1α, but not HIF-2α, was essential for the effector state in CD8+ T cells. Furthermore, loss of HIF-1α in CD8+ T cells reduced tumor infiltration and tumor cell killing, and altered tumor vascularization. Deletion of VEGF-A, an HIF target gene, in CD8+ T cells accelerated tumorigenesis while also altering vascularization. Analyses of human breast cancer showed inverse correlations between VEGF-A expression and CD8+ T cell infiltration, and a link between T cell infiltration and vascularization. These data demonstrate that the HIF-1α/VEGF-A axis is an essential aspect of tumor immunity.

CD69 is a direct HIF-1α target gene in hypoxia as a mechanism enhancing expression on tumor-infiltrating T lymphocytes

ABSTRACT CD69 is an early activation marker on the surface of T lymphocytes undergoing activation by cognate antigen. We observed intense expression of CD69 on tumor-infiltrating T-lymphocytes that reside in the hypoxic tumor microenvironment and hypothesized that CD69 could be, at least partially, under the control of the transcriptional hypoxia response. In line with this, human and mouse CD3-stimulated lymphocytes cultured under hypoxia (1% O2) showed increased expression of CD69 at the protein and mRNA level. Consistent with these findings, mouse T lymphocytes that had recently undergone hypoxia in vivo, as denoted by pimonidazole staining, were more frequently CD69+ in the tumor and bone marrow hypoxic tissue compartments. We found evidence for HIF-1α involvement both when using T-lymphocytes from inducible HIF-1α−/− mice and when observing tumor-infiltrating T-lymphocytes in mice whose T cells are HIF-1α−/−. Direct pro-transcriptional activity of HIF-1α on a newly identified hypoxia response element (HRE) found in the human CD69 locus was demonstrated by ChIP experiments. These results uncover a connection between the HIF-1α oxygen-sensing pathway and CD69 immunobiology.

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia

Summary Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.

Modelling pulmonary microthrombosis coupled to metastasis: distinct effects of thrombogenesis on tumorigenesis

ABSTRACT Thrombosis can cause localized ischemia and tissue hypoxia, and both of these are linked to cancer metastasis. Vascular micro-occlusion can occur as a result of arrest of circulating tumour cells in small capillaries, giving rise to microthrombotic events that affect flow, creating localized hypoxic regions. To better understand the association between metastasis and thrombotic events, we generated an experimental strategy whereby we modelled the effect of microvascular occlusion in metastatic efficiency by using inert microbeads to obstruct lung microvasculature before, during and after intravenous tumour cell injection. We found that controlled induction of a specific number of these microthrombotic insults in the lungs caused an increase in expression of the hypoxia-inducible transcription factors (HIFs), a pro-angiogenic and pro-tumorigenic environment, as well as an increase in myeloid cell infiltration. Induction of pulmonary microthrombosis prior to introduction of tumour cells to the lungs had no effect on tumorigenic success, but thrombosis at the time of tumour cell seeding increased number and size of tumours in the lung, and this effect was strikingly more pronounced when the micro-occlusion occurred on the day following introduction of tumour cells. The tumorigenic effect of microbead treatment was seen even when thrombosis was induced five days after tumour cell injection. We also found positive correlations between thrombotic factors and expression of HIF2α in human tumours. The model system described here demonstrates the importance of thrombotic insult in metastatic success and can be used to improve understanding of thrombosis-associated tumorigenesis and its treatment. Summary: Induction of pulmonary microthrombosis by three distinct methods enhances HIF-a expression and tumour formation; increases in tumorigenesis that are induced by these thrombotic insults occur in a time- and mode-dependent manner.

Abstract A19: Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors

Thrombosis causes local blood flow restriction and tissue hypoxia, and both of these are associated with tumor cell metastasis. To better understand the regulation of thrombosis-induced metastasis, we created a model incorporating elements of both processes. Controlled induction of pulmonary microthrombosis caused an increase in expression of first hypoxia-inducible factor (HIF)1α, and subsequently HIF2α. Induction of thrombosis before the introduction of tumor cells to venous circulation had no effect on pulmonary tumor number or size; but thrombosis at the time of tumor cell seeding increased number and size of tumors in the lung. Thrombosis on the day after seeding of tumor cells caused an even greater increase in tumor number and size, and this effect persisted until even when thrombosis was induced five days after the introduction of tumor cells into the blood. Experiments on myeloid HIF1α or HIF2α knockout mice demonstrated that loss of either HIF1α or HIF2α eliminated the advantage given to pulmonary tumorigenesis by thrombotic insult. In primary human tumours, markers of thrombosis were positively correlated with expression of the 2 HIFα isoforms. These data demonstrate the importance of microthrombosis in a novel model of metastasis and the essential role of the myeloid cell-specific HIFα response in mediating this process. Citation Format: Colin E. Evans, Asis Palazon, Jingwei Sim, Petros A. Tyrakis, Par-Ola Bendahl, Mattias Belting, Helene Rundqvist, Cristina Branco, Randall S. Johnson. Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A19.

Abstract B12: Molecular mechanisms of HIF-1a and HIF-2a activation in nonmalignant cells at the premetastatic niche: Effects on metastatic success

Tumor hypoxia and the hypoxia-inducible transcription factors (HIF) have been thoroughly characterized in solid tumors, and they are active participants in pathways promoting tumor growth and dispersion. However, despite their well-known pro-tumorigenic role when stabilized in cancer cells, HIF activation in other cell types has been shown to be less consistent and often antagonistic. We have previously shown that specific deletion of HIF-1a in endothelial cells results in reduced tumor cell migration and metastatic incidence, whereas deletion of HIF-2a in this same cell type has the opposite effect. This phenomenon also involved endothelial-specific nitric oxide production via the inducible NOS (iNOS). Endothelial cells are at the interface between the primary tumor and the rest of the body, and play a role not only in tumor maintenance and growth, but also in metastatic success and proliferation at secondary sites. Immune/inflammatory cells are also known to be active players in tumor cell motility and establishment. We are interested in understanding the role of non-malignant cell types in the metastatic process, specifically (i) signals that trigger HIF-1a or HIF-2a activation at metastatic sites and (ii) how does the stabilization of either HIF isoform (or both) affect tumor cell recruitment and establishment. This work uses primary cells isolated from different genotypic backgrounds to dissect the hypoxia-response regulatory pathways, using a combination of screenings: transcription factor activation and downstream transcriptional targets, in order to build a map of HIF-dependent metabolism. Specifically, we are interested in the pathways which activation or silencing could have an effect on vascular morphology, permeability, inter-cellular signaling and post-extravasation events. Complementary studies in physiological models of extravasation were used for histological, molecular and biochemical analysis of hypoxic tissue to study molecular players identified by our initial ex-vivo screening. Citation Format: Cristina Branco-Price, Asis Palazon, Jessica Xie, Randall S. Johnson. Molecular mechanisms of HIF-1a and HIF-2a activation in nonmalignant cells at the premetastatic niche: Effects on metastatic success. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B12. doi:10.1158/1538-7445.CHTME14-B12