Orphan Nuclear Receptor Family 4A (NR4A) Members NR4A2 and NR4A3 Selectively Modulate Elements of the Monocyte Response to Buffered Hypercapnia.

Hypercapnia occurs when the partial pressure of carbon dioxide (CO2) in the blood exceeds 45 mmHg. Hypercapnia is associated with several lung pathologies and is transcriptionally linked to suppression of immune and inflammatory signalling through poorly understood mechanisms. Here we propose Orphan Nuclear Receptor Family 4A (NR4A) family members NR4A2 and NR4A3 as potential transcriptional regulators of the cellular response to hypercapnia in monocytes. Using a THP-1 monocyte model, we investigated the sensitivity of NR4A family members to CO2 and the impact of depleting NR4A2 and NR4A3 on the monocyte response to buffered hypercapnia (10% CO2) using RNA-sequencing. We observed that NR4A2 and NR4A3 are CO2-sensitive transcription factors and that depletion of NR4A2 and NR4A3 led to reduced CO2-sensitivity of mitochondrial and heat shock protein (Hsp)-related genes, respectively. Several CO2-sensitive genes were, however, refractory to depletion of NR4A2 and NR4A3, indicating that NR4As regulate certain elements of the cellular response to buffered hypercapnia but that other transcription factors also contribute. Bioinformatic analysis of conserved CO2-sensitive genes implicated several novel putative CO2-sensitive transcription factors, of which the ETS Proto-Oncogene 1 Transcription Factor (ETS-1) was validated to show increased nuclear expression in buffered hypercapnia. These data give significant insights into the understanding of immune responses in patients experiencing hypercapnia.

Hypercapnia alters mitochondrial gene expression and acylcarnitine production in monocytes.

CO2 is produced during aerobic respiration. Normally, levels of CO2 in the blood are tightly regulated but pCO2 can rise (hypercapnia, pCO2 > 45 mmHg) in patients with lung diseases, for example, chronic obstructive pulmonary disease (COPD). Hypercapnia is a risk factor in COPD but may be of benefit in the context of destructive inflammation. The effects of CO2 per se, on transcription, independent of pH change are poorly understood and warrant further investigation. Here we elucidate the influence of hypercapnia on monocytes and macrophages through integration of state-of-the-art RNA-sequencing, metabolic and metabolomic approaches. THP-1 monocytes and interleukin 4-polarized primary murine macrophages were exposed to 5% CO2 versus 10% CO2 for up to 24 h in pH-buffered conditions. In hypercapnia, we identified around 370 differentially expressed genes (DEGs) under basal and about 1889 DEGs under lipopolysaccharide-stimulated conditions in monocytes. Transcripts relating to both mitochondrial and nuclear-encoded gene expression were enhanced in hypercapnia in basal and lipopolysaccharide-stimulated cells. Mitochondrial DNA content was not enhanced, but acylcarnitine species and genes associated with fatty acid metabolism were increased in hypercapnia. Primary macrophages exposed to hypercapnia also increased activation of genes associated with fatty acid metabolism and reduced activation of genes associated with glycolysis. Thus, hypercapnia elicits metabolic shifts in lipid metabolism in monocytes and macrophages under pH-buffered conditions. These data indicate that CO2 is an important modulator of monocyte transcription that can influence immunometabolic signaling in immune cells in hypercapnia. These immunometabolic insights may be of benefit in the treatment of patients experiencing hypercapnia.

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.

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.

Transcriptional Profiling of Monocytes Deficient in Nuclear Orphan Receptors NR4A2 and NR4A3 Reveals Distinct Signalling Roles Related to Antigen Presentation and Viral Response.

The nuclear receptor sub-family 4 group A (NR4A) family are early response genes that encode proteins that are activated in several tissues/cells in response to a variety of stressors. The NR4A family comprises NR4A1, NR4A2 and NR4A3 of which NR4A2 and NR4A3 are under researched and less understood, particularly in the context of immune cells. NR4A expression is associated with multiple diseases e.g. arthritis and atherosclerosis and the development of NR4A-targetting molecules as therapeutics is a current focus in this research field. Here, we use a combination of RNA-sequencing coupled with strategic bioinformatic analysis to investigate the down-stream effects of NR4A2 and NR4A3 in monocytes and dissect their common and distinct signalling roles. Our data reveals that NR4A2 and NR4A3 depletion has a robust and broad-reaching effect on transcription in both the unstimulated state and in the presence of LPS. Interestingly, many of the genes affected were present in both the unstimulated and stimulated states revealing a previously unappreciated role for the NR4As in unstimulated cells. Strategic clustering and bioinformatic analysis identified both distinct and common transcriptional roles for NR4A2 and NR4A3 in monocytes. NR4A2 notably was linked by both bioinformatic clustering analysis and transcription factor interactome analysis to pathways associated with antigen presentation and regulation of MHC genes. NR4A3 in contrast was more closely linked to pathways associated with viral response. Functional studies further support our data analysis pointing towards preferential/selective roles for NR4A2 in the regulation of antigen processing with common roles for NR4A2 and NR4A3 evident with respect to cell migration. Taken together this study provides novel mechanistic insights into the role of the enigmatic nuclear receptors NR4A2 and NR4A3 in monocytes.

The NR4A agonist, Cytosporone B, attenuates pro-inflammatory mediators in human colorectal cancer tissue ex vivo.

Inflammation is a pivotal pathological factor in colorectal cancer (CRC) initiation and progression, and modulating this inflammatory state has the potential to ameliorate disease progression. NR4A receptors have emerged as key regulators of inflammatory pathways that are important in CRC. Here, we have examined the effect of NR4A agonist, Cytosporone B (CsnB), on colorectal tissue integrity and its effect on the inflammatory profile in CRC tissue ex vivo. Here, we demonstrate concentrations up 100 µM CsnB did not adversely affect tissue integrity as measured using transepithelial electrical resistance, histology and crypt height. Subsequently, we reveal through the use of a cytokine/chemokine array, ELISA and qRT-PCR analysis that multiple pro-inflammatory mediators were significantly increased in CRC tissue compared to control tissue, which were then attenuated with the addition of CsnB (such as IL-1ß, IL-8 and TNFα). Lastly, stratification of the data revealed that CsnB especially alters the inflammatory profile of tumours derived from males who had not undergone chemoradiotherapy. Thus, this study demonstrates that NR4A agonist CsnB does not adversely affect colon tissue structure or functionality and can attenuate the pro-inflammatory state of human CRC tissue ex vivo.

Protein kinase D, ubiquitin and proteasome pathways are involved in adenosine receptor-stimulated NR4A expression in myeloid cells.

Adenosine is a purine nucleoside pivotal for homeostasis in cells and tissues. Stimulation of the adenosine receptors (AR) has been shown to regulate the nuclear orphan receptor 4A (NR4A1-3) family, resulting in attenuation of hyper-inflammatory responses in myeloid cells. The NR4A1-3 orphan receptors are early immediate response genes and transcriptional regulators of cell and tissue homeostasis. The signal transduction and transcriptional mechanism(s) of how AR-stimulation promotes NR4A expression in myeloid cells is unknown and is the focus of this study. We confirm that adenosine and the stable analogue, 5'-N-Ethylcarboxamidoadenosine (NECA), enhance NR4A1-3 expression in THP-1 cells. Pharmacological approaches identified that protein kinase D (PKD) mediates AR-stimulated NR4A expression in myeloid cells and reveals no involvement of PKA nor PKC. The role of NF-κB, a principal regulator of NR4A expression in myeloid cells, was examined as a possible transcriptional regulator downstream of PKD. Utilising BAY11-7082 and MG-132, inhibitors of the respective ubiquitin and proteasome pathways essential for NF-κB activation, suggested a prospective role for NF-κB, or more specifically signalling via IKKα/ß. However, biological interventional studies using overexpression of IκBα in myeloid cells and MEF cells lacking IKKα and IKKß (IKKα/ß-/-) revealed the NF-κB pathway is not utilised in mediating AR-stimulated NR4A expression. Thus, this study contributes mechanistic insight into how AR signalling modulates the expression of NR4A receptors, pivotal regulators of inflammatory responses in myeloid cells.

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.

Understanding the pathophysiological mechanisms of cardiometabolic complications in obstructive sleep apnoea: towards personalised treatment approaches.

In January 2019, a European Respiratory Society research seminar entitled "Targeting the detrimental effects of sleep disturbances and disorders" was held in Dublin, Ireland. It provided the opportunity to critically review the current evidence of pathophysiological responses of sleep disturbances, such as sleep deprivation, sleep fragmentation or circadian misalignment and of abnormalities in physiological gases such as oxygen and carbon dioxide, which occur frequently in respiratory conditions during sleep. A specific emphasis of the seminar was placed on the evaluation of the current state of knowledge of the pathophysiology of cardiovascular and metabolic diseases in obstructive sleep apnoea (OSA). Identification of the detailed mechanisms of these processes is of major importance to the field and this seminar offered an ideal platform to exchange knowledge, and to discuss pitfalls of current models and the design of future collaborative studies. In addition, we debated the limitations of current treatment strategies for cardiometabolic complications in OSA and discussed potentially valuable alternative approaches.

Mechanisms and Consequences of Oxygen and Carbon Dioxide Sensing in Mammals.

Molecular oxygen (O2) and carbon dioxide (CO2) are the primary gaseous substrate and product of oxidative phosphorylation in respiring organisms, respectively. Variance in the levels of either of these gasses outside of the physiological range presents a serious threat to cell, tissue, and organism survival. Therefore, it is essential that endogenous levels are monitored and kept at appropriate concentrations to maintain a state of homeostasis. Higher organisms such as mammals have evolved mechanisms to sense O2 and CO2 both in the circulation and in individual cells and elicit appropriate corrective responses to promote adaptation to commonly encountered conditions such as hypoxia and hypercapnia. These can be acute and transient nontranscriptional responses, which typically occur at the level of whole animal physiology or more sustained transcriptional responses, which promote chronic adaptation. In this review, we discuss the mechanisms by which mammals sense changes in O2 and CO2 and elicit adaptive responses to maintain homeostasis. We also discuss crosstalk between these pathways and how they may represent targets for therapeutic intervention in a range of pathological states.

Elevated CO2 regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans.

Carbon dioxide (CO2) is sensed by cells and can trigger signals to modify gene expression in different tissues leading to changes in organismal functions. Despite accumulating evidence that several pathways in various organisms are responsive to CO2 elevation (hypercapnia), it has yet to be elucidated how hypercapnia activates genes and signaling pathways, or whether they interact, are integrated, or are conserved across species. Here, we performed a large-scale transcriptomic study to explore the interaction/integration/conservation of hypercapnia-induced genomic responses in mammals (mice and humans) as well as invertebrates (Caenorhabditis elegans and Drosophila melanogaster). We found that hypercapnia activated genes that regulate Wnt signaling in mouse lungs and skeletal muscles in vivo and in several cell lines of different tissue origin. Hypercapnia-responsive Wnt pathway homologues were similarly observed in secondary analysis of available transcriptomic datasets of hypercapnia in a human bronchial cell line, flies and nematodes. Our data suggest the evolutionarily conserved role of high CO2 in regulating Wnt pathway genes.

Protein Hydroxylation by Hypoxia-Inducible Factor (HIF) Hydroxylases: Unique or Ubiquitous?

All metazoans that utilize molecular oxygen (O2) for metabolic purposes have the capacity to adapt to hypoxia, the condition that arises when O2 demand exceeds supply. This is mediated through activation of the hypoxia-inducible factor (HIF) pathway. At physiological oxygen levels (normoxia), HIF-prolyl hydroxylases (PHDs) hydroxylate proline residues on HIF-α subunits leading to their destabilization by promoting ubiquitination by the von-Hippel Lindau (VHL) ubiquitin ligase and subsequent proteasomal degradation. HIF-α transactivation is also repressed in an O2-dependent way due to asparaginyl hydroxylation by the factor-inhibiting HIF (FIH). In hypoxia, the O2-dependent hydroxylation of HIF-α subunits by PHDs and FIH is reduced, resulting in HIF-α accumulation, dimerization with HIF-ß and migration into the nucleus to induce an adaptive transcriptional response. Although HIFs are the canonical substrates for PHD- and FIH-mediated protein hydroxylation, increasing evidence indicates that these hydroxylases may also have alternative targets. In addition to PHD-conferred alterations in protein stability, there is now evidence that hydroxylation can affect protein activity and protein/protein interactions for alternative substrates. PHDs can be pharmacologically inhibited by a new class of drugs termed prolyl hydroxylase inhibitors which have recently been approved for the treatment of anemia associated with chronic kidney disease. The identification of alternative targets of HIF hydroxylases is important in order to fully elucidate the pharmacology of hydroxylase inhibitors (PHI). Despite significant technical advances, screening, detection and verification of alternative functional targets for PHDs and FIH remain challenging. In this review, we discuss recently proposed non-HIF targets for PHDs and FIH and provide an overview of the techniques used to identify these.

Hydroxylase Inhibition Selectively Induces Cell Death in Monocytes.

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1ß-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1ß. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.

Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.

CO2 is a physiological gas normally produced in the body during aerobic respiration. Hypercapnia (elevated blood pCO2 >≈50 mm Hg) is a feature of several lung pathologies, e.g. chronic obstructive pulmonary disease. Hypercapnia is associated with increased susceptibility to bacterial infections and suppression of inflammatory signaling. The NF-κB pathway has been implicated in these effects; however, the molecular mechanisms underpinning cellular sensitivity of the NF-κB pathway to CO2 are not fully elucidated. Here, we identify several novel CO2-dependent changes in the NF-κB pathway. NF-κB family members p100 and RelB translocate to the nucleus in response to CO2 A cohort of RelB protein-protein interactions (e.g. with Raf-1 and IκBα) are altered by CO2 exposure, although others are maintained (e.g. with p100). RelB is processed by CO2 in a manner dependent on a key C-terminal domain located in its transactivation domain. Loss of the RelB transactivation domain alters NF-κB-dependent transcriptional activity, and loss of p100 alters sensitivity of RelB to CO2 Thus, we provide molecular insight into the CO2 sensitivity of the NF-κB pathway and implicate altered RelB/p100-dependent signaling in the CO2-dependent regulation of inflammatory signaling.

NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells.

Dysregulation of inflammatory responses is a hallmark of multiple diseases such as atherosclerosis and rheumatoid arthritis. As constitutively active transcription factors, NR4A nuclear receptors function to control the magnitude of inflammatory responses and in chronic inflammatory disease can be protective or pathogenic. Within this study, we demonstrate that TLR4 stimulation using the endotoxin lipopolysaccharide (LPS) rapidly enhances NR4A1-3 expression in human and murine, primary and immortalized myeloid cells with concomitant gene transcription and protein secretion of MIP-3α, a central chemokine implicated in numerous pathologies. Deficiency of NR4A2 and NR4A3 in human and murine myeloid cells reveals that both receptors function as positive regulators of enhanced MIP-3α expression. In contrast, within the same cell types and conditions, altered NR4A activity leads to suppression of LPS-induced MCP-1 gene and protein expression. An equivalent pattern of inflammatory gene regulation is replicated in TNFα-treated myeloid cells. We show that NF-κB is the critical regulator of NR4A1-3, MIP-3α, and MCP-1 during TLR4 stimulation in myeloid cells and highlight a parallel mechanism whereby NR4A activity can repress or enhance NF-κB target gene expression simultaneously. Mechanistic insight reveals that NR4A2 does not require DNA-binding capacity in order to enhance or repress NF-κB target gene expression simultaneously and establishes a role for NF-κB family member Relb as a novel NR4A target gene involved in the positive regulation of MIP-3α. Thus, our data reveal a dynamic role for NR4A receptors concurrently enhancing and repressing NF-κB activity in myeloid cells leading to altered transcription of key inflammatory mediators.

Hypoxia and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a general term to describe inflammatory diseases of the gastrointestinal tract such as Crohn's disease and ulcerative colitis. IBD affects approximately 1 in 200 individuals and exerts a significant health and quality of life burden on patients. Surgical intervention can be curative in ulcerative colitis but there is currently no cure for Crohn's disease. Since this is the case, and the fact that patients are often diagnosed at a young age, IBD exerts a significant financial burden on the health care system, and society as a whole. The underlying pathology of IBD is complex and involves a combination of genetic, environmental and microbial factors. Regardless of the underlying causes of the condition, this disease is universally characterized by disruption to the protective epithelial barrier separating the intestinal lumen above from the mucosal immune system below. Once this barrier becomes compromised a sequence of events ensues, that can occur in repetitive cycles to ensure long-term and serious damage to the gut. The role of hypoxia and hypoxia-dependent signalling pathways are increasingly appreciated to play a role in the physiology and pathophysiology of the intestine. The intestinal epithelium normally exists in a state of physiological hypoxia, with additional tissue hypoxia a feature of active inflammatory disease. Furthermore, recent pre-clinical animal studies have clearly supported the rationale for pharmacologically manipulating the oxygen-sensitive hypoxia-inducible factor (HIF) pathway in models of IBD. Thus, this review will discuss the contribution of hypoxia sensitive pathways in the pathology of IBD. Finally we will discuss the emerging evidence for manipulation of hypoxia-sensitive pathways in the treatment of IBD.

Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion.

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.

Hydroxylase inhibition regulates inflammation-induced intestinal fibrosis through the suppression of ERK-mediated TGF-ß1 signaling. [corrected].

Fibrosis is a complication of chronic inflammatory disorders such as inflammatory bowel disease, a condition which has limited therapeutic options and often requires surgical intervention. Pharmacologic inhibition of oxygen-sensing prolyl hydroxylases, which confer oxygen sensitivity upon the hypoxia-inducible factor pathway, has recently been shown to have therapeutic potential in colitis, although the mechanisms involved remain unclear. Here, we investigated the impact of hydroxylase inhibition on inflammation-driven fibrosis in a murine colitis model. Mice exposed to dextran sodium sulfate, followed by a period of recovery, developed intestinal fibrosis characterized by alterations in the pattern of collagen deposition and infiltration of activated fibroblasts. Treatment with the hydroxylase inhibitor dimethyloxalylglycine ameliorated fibrosis. TGF-ß1 is a key regulator of fibrosis that acts through the activation of fibroblasts. Hydroxylase inhibition reduced TGF-ß1-induced expression of fibrotic markers in cultured fibroblasts, suggesting a direct role for hydroxylases in TGF-ß1 signaling. This was at least in part due to inhibition of noncanonical activation of extracellular signal-regulated kinase (ERK) signaling. In summary, pharmacologic hydroxylase inhibition ameliorates intestinal fibrosis through suppression of TGF-ß1-dependent ERK activation in fibroblasts. We hypothesize that in addition to previously reported immunosupressive effects, hydroxylase inhibitors independently suppress profibrotic pathways.