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.

Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition.

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFß) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFß. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

Mucosal inflammation downregulates PHD1 expression promoting a barrier-protective HIF-1α response in ulcerative colitis patients.

The HIF hydroxylase enzymes (PHD1-3 and FIH) are cellular oxygen-sensors which confer hypoxic-sensitivity upon the hypoxia-inducible factors HIF-1α and HIF-2α. Microenvironmental hypoxia has a strong influence on the epithelial and immune cell function through HIF-dependent gene expression and consequently impacts upon the course of disease progression in ulcerative colitis (UC), with HIF-1α being protective while HIF-2α promotes disease. However, little is known about how inflammation regulates hypoxia-responsive pathways in UC patients. Here we demonstrate that hypoxia is a prominent microenvironmental feature of the mucosa in UC patients with active inflammatory disease. Furthermore, we found that inflammation drives transcriptional programming of the HIF pathway including downregulation of PHD1 thereby increasing the tissue responsiveness to hypoxia and skewing this response toward protective HIF-1 over detrimental HIF-2 activation. We identified CEBPα as a transcriptional regulator of PHD1 mRNA expression which is downregulated in both inflamed tissue derived from patients and in cultured intestinal epithelial cells treated with inflammatory cytokines. In summary, we propose that PHD1 downregulation skews the hypoxic response toward enhanced protective HIF-1α stabilization in the inflamed mucosa of UC patients.

Pharmacologic inhibition of hypoxia-inducible factor (HIF)-hydroxylases ameliorates allergic contact dermatitis.

BACKGROUND: When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway. METHODS: Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development. RESULTS: Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway. CONCLUSION: These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.

Role of hypoxia-inducible factor 1 in type 1 diabetes.

Type 1 diabetes (T1D) is a common autoimmune disease in which dysregulated glucose metabolism is a key feature. T1D is both poorly understood and in need of improved therapeutics. Hypoxia is frequently encountered in multiple tissues in T1D patients including the pancreas and sites of diabetic complications. Hypoxia-inducible factor (HIF)-1, a ubiquitous master regulator of the adaptive response to hypoxia, promotes glucose metabolism through transcriptional and non-transcriptional mechanisms and alters disease progression in multiple preclinical T1D models. However, how HIF-1 activation in ß-cells of the pancreas and immune cells (two key cell types in T1D) ultimately affects disease progression remains controversial. We discuss recent advances in our understanding of the role of hypoxia/HIF-1-induced glycolysis in T1D and explore the possible use of drugs targeting this pathway as potential new therapeutics.

Beyond butyrate: microbial fiber metabolism supporting colonic epithelial homeostasis.

Human gut bacteria produce metabolites that support energy and carbon metabolism of colonic epithelial cells. While butyrate is commonly considered the primary fuel, it alone cannot meet all the carbon requirements for cellular synthetic functions. Glucose, delivered via circulation or microbial metabolism, serves as a universal carbon source for synthetic processes like DNA, RNA, protein, and lipid production. Detailed knowledge of epithelial carbon and energy metabolism is particularly relevant for epithelial regeneration in digestive and metabolic diseases, such as inflammatory bowel disease and type 2 diabetes. Here, we review the production and role of different colonic microbial metabolites in energy and carbon metabolism of colonocytes, also critically evaluating the common perception that butyrate is the preferred fuel.

Extracellular vesicles derived from stressed beta cells mediate monocyte activation and contribute to islet inflammation.

Objective: Beta cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In recent years, the role played by beta cells in the development of T1D has evolved from passive victims of the immune system to active contributors in their own destruction. We and others have demonstrated that perturbations in the islet microenvironment promote endoplasmic reticulum (ER) stress in beta cells, leading to enhanced immunogenicity. Among the underlying mechanisms, secretion of extracellular vesicles (EVs) by beta cells has been suggested to mediate the crosstalk with the immune cell compartment. Methods: To study the role of cellular stress in the early events of T1D development, we generated a novel cellular model for constitutive ER stress by modulating the expression of HSPA5, which encodes BiP/GRP78, in EndoC-ßH1 cells. To investigate the role of EVs in the interaction between beta cells and the immune system, we characterized the EV miRNA cargo and evaluated their effect on innate immune cells. Results: Analysis of the transcriptome showed that HSPA5 knockdown resulted in the upregulation of signaling pathways involved in the unfolded protein response (UPR) and changes the miRNA content of EVs, including reduced levels of miRNAs involved in IL-1ß signaling. Treatment of primary human monocytes with EVs from stressed beta cells resulted in increased surface expression of CD11b, HLA-DR, CD40 and CD86 and upregulation of IL-1ß and IL-6. Conclusion: These findings indicate that the content of EVs derived from stressed beta cells can be a mediator of islet inflammation.

The NRF2/Keap1 pathway as a therapeutic target in inflammatory bowel disease.

Oxidative stress (OS) is an important pathophysiological mechanism in inflammatory bowel disease (IBD). However, clinical trials investigating compounds directly targeting OS in IBD yielded mixed results. The NRF2 (nuclear factor erythroid 2-related factor 2)/Keap1 (Kelch-like ECH-associated protein 1) pathway orchestrates cellular responses to OS, and dysregulation of this pathway has been implicated in IBD. Activation of the NRF2/Keap1 pathway may enhance antioxidant responses. Although this approach could help to attenuate OS and potentially improve clinical outcomes, an overview of human evidence for modulating the NRF2/Keap1 axis and more recent developments in IBD is lacking. This review explores the NRF2/Keap1 pathway as potential therapeutic target in IBD and presents compounds activating this pathway for future clinical applications.

Faecalibacterium prausnitzii promotes intestinal epithelial IL-18 production through activation of the HIF1α pathway.

Introduction: Intestinal epithelial cells produce interleukin-18 (IL-18), a key factor in promoting epithelial barrier integrity. Here, we analyzed the potential role of gut bacteria and the hypoxia-inducible factor 1α (HIF1α) pathway in regulating mucosal IL18 expression in inflammatory bowel disease (IBD). Methods: Mucosal samples from patients with IBD (n = 760) were analyzed for bacterial composition, IL18 levels and HIF1α pathway activation. Wild-type Caco-2 and CRISPR/Cas9-engineered Caco-2-HIF1A-null cells were cocultured with Faecalibacterium prausnitzii in a "Human oxygen-Bacteria anaerobic" in vitro system and analyzed by RNA sequencing. Results: Mucosal IL18 mRNA levels correlated positively with the abundance of mucosal-associated butyrate-producing bacteria, in particular F. prausnitzii, and with HIF1α pathway activation in patients with IBD. HIF1α-mediated expression of IL18, either by a pharmacological agonist (dimethyloxallyl glycine) or F. prausnitzii, was abrogated in Caco-2-HIF1A-null cells. Conclusion: Butyrate-producing gut bacteria like F. prausnitzii regulate mucosal IL18 expression in a HIF1α-dependent manner that may aid in mucosal healing in IBD.

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.

Inulin-grown Faecalibacterium prausnitzii cross-feeds fructose to the human intestinal epithelium.

Many chronic diseases are associated with decreased abundance of the gut commensal Faecalibacterium prausnitzii. This strict anaerobe can grow on dietary fibers, e.g., prebiotics, and produce high levels of butyrate, often associated to epithelial metabolism and health. However, little is known about other F. prausnitzii metabolites that may affect the colonic epithelium. Here, we analyzed prebiotic cross-feeding between F. prausnitzii and intestinal epithelial (Caco-2) cells in a "Human-oxygen Bacteria-anaerobic" coculture system. Inulin-grown F. prausnitzii enhanced Caco-2 viability and suppressed inflammation- and oxidative stress-marker expression. Inulin-grown F. prausnitzii produced excess butyrate and fructose, but only fructose efficiently promoted Caco-2 growth. Finally, fecal microbial taxonomy analysis (16S sequencing) from healthy volunteers (n = 255) showed the strongest positive correlation for F. prausnitzii abundance and stool fructose levels. We show that fructose, produced and accumulated in a fiber-rich colonic environment, supports colonic epithelium growth, while butyrate does not.

Determinants of hypoxia-inducible factor activity in the intestinal mucosa.

The intestinal mucosa is exposed to fluctuations in oxygen levels due to constantly changing rates of oxygen demand and supply and its juxtaposition with the anoxic environment of the intestinal lumen. This frequently results in a state of hypoxia in the healthy mucosa even in the physiologic state. Furthermore, pathophysiologic hypoxia (which is more severe and extensive) is associated with chronic inflammatory diseases including inflammatory bowel disease (IBD). The hypoxia-inducible factor (HIF), a ubiquitously expressed regulator of cellular adaptation to hypoxia, is central to both the adaptive and the inflammatory responses of cells of the intestinal mucosa in IBD patients. In this review, we discuss the microenvironmental factors which influence the level of HIF activity in healthy and inflamed intestinal mucosae and the consequences that increased HIF activity has for tissue function and disease progression.