Stefan F. Ehrentraut

Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function

Interactions between the microbiota and distal gut are fundamental determinants of human health. Such interactions are concentrated at the colonic mucosa and provide energy for the host epithelium through the production of the short-chain fatty acid butyrate. We sought to determine the role of epithelial butyrate metabolism in establishing the austere oxygenation profile of the distal gut. Bacteria-derived butyrate affects epithelial O2 consumption and results in stabilization of hypoxia-inducible factor (HIF), a transcription factor coordinating barrier protection. Antibiotic-mediated depletion of the microbiota reduces colonic butyrate and HIF expression, both of which are restored by butyrate supplementation. Additionally, germ-free mice exhibit diminished retention of O2-sensitive dyes and decreased stabilized HIF. Furthermore, the influences of butyrate are lost in cells lacking HIF, thus linking butyrate metabolism to stabilized HIF and barrier function. This work highlights a mechanism where host-microbe interactions augment barrier function in the distal gut.

CD73+ regulatory T cells contribute to adenosine-mediated resolution of acute lung injury

Acute lung injury (ALI) is characterized by alveolar injury and uncontrolled inflammation. Since most cases of ALI resolve spontaneously, understanding the endogenous mechanisms that promote ALI resolution is important to developing effective therapies. Previous studies have implicated extracellular adenosine signaling in tissue adaptation and wound healing. Therefore, we hypothesized a functional contribution for the endogenous production of adenosine during ALI resolution. As a model, we administered intratracheal LPS and observed peak lung injury at 3 d, with resolution by d 14. Treatment with pegylated adenosine‐deaminase to enhance extracellular adenosine breakdown revealed impaired ALI resolution. Similarly, genetic deletion of cd73, the pacemaker for extracellular adenosine generation, was associated with increased mortality (0% wild‐type and 40% in cd73–/– mice; P<0.05) and failure to resolve ALI adequately. Studies of inflammatory cell trafficking into the lungs during ALI resolution revealed that regulatory T cells (Tregs) express the highest levels of CD73. While Treg numbers in cd73–/– mice were similar to controls, cd73‐deficient Tregs had attenuated immunosuppressive functions. Moreover, adoptive transfer of cd73‐deficient Tregs into Rag–/– mice emulated the observed phenotype in cd73–/– mice, while transfer of wild‐type Tregs was associated with normal ALI resolution. Together, these studies implicate CD73‐dependent adenosine generation in Tregs in promoting ALI resolution.—Ehrentraut, H., Clambey, E. T., McNamee, E. N., Brodsky, K. S., Ehrentraut, S. F., Poth, J. M., Riegel, A. K., Westrich, J. A., Colgan, S. P., Eltzschig, H. K. CD73+ regulatory T cells contribute to adenosine‐mediated resolution of acute lung injury. FASEB J. 27, 2207–2219 (2013). www.fasebj.org

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis

Significance Intestinal epithelial barrier dysregulation is a hallmark of inflammatory bowel diseases (IBDs). A central role for hypoxic signaling has been defined in barrier modulation during inflammation. We demonstrate that genes involved in creatine metabolism, the creatine kinases (CKs), are coordinately regulated by hypoxia-inducible transcription factors (HIFs) and that such regulation is critical to barrier function. Inhibition of the CK pathway abrogates apical junction assembly and barrier integrity. Dietary creatine supplementation profoundly attenuates the pathogenic course of mucosal inflammation in mouse colitis models. Moreover, we demonstrate altered expression of mitochondrial and cytosolic CK enzymes in IBD patient tissue. These findings highlight the fundamental contribution of creatine metabolism to intestinal mucosal function, homeostasis, and disease resolution. Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine–creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2–dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.

IFN-γ–Mediated Induction of an Apical IL-10 Receptor on Polarized Intestinal Epithelia

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1–null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion

Ischemia and reperfusion‐elicited tissue injury contributes to morbidity and mortality of hepatic surgery and during liver transplantation. Previous studies implicated extracellular adenosine signaling in liver protection. Based on the notion that extracellular adenosine signaling is terminated by uptake from the extracellular towards the intracellular compartment by way of equilibrative nucleoside transporters (ENTs), we hypothesized a functional role of ENTs in liver protection from ischemia. During orthotopic liver transplantation in humans, we observed higher expressional levels of ENT1 than ENT2, in conjunction with repression of ENT1 and ENT2 transcript and protein levels following warm ischemia and reperfusion. Treatment with the pharmacologic ENT inhibitor dipyridamole revealed elevations of hepatic adenosine levels and robust liver protection in a murine model of liver ischemia and reperfusion. Studies in gene‐targeted mice for Ent1 or Ent2 demonstrated selective protection from liver injury in Ent1−/− mice. Treatment with selective adenosine receptor antagonists indicated a contribution of Adora2b receptor signaling in ENT‐dependent liver protection. Conclusion: These findings implicate ENT1 in liver protection from ischemia and reperfusion injury and suggest ENT inhibitors may be of benefit in the prevention or treatment of ischemic liver injury. (Hepatology 2013;58:1766–1778)

IN VIVO TOLL-LIKE RECEPTOR 4 ANTAGONISM RESTORES CARDIAC FUNCTION DURING ENDOTOXEMIA

ABSTRACT Severe sepsis and septic shock are often accompanied by acute cardiovascular depression. Lipopolysaccharide (LPS) signaling via Toll-like receptor 4 (TLR4) can induce septic organ dysfunction. The aim of this study was to elucidate the in vivo impact of pharmacological TLR4 antagonism on LPS-induced cardiovascular depression using eritoran tetrasodium (E5564). To simulate sepsis, C3H/HeN mice were challenged i.p. with 2 mg/kg body weight LPS. With the intent to antagonize the LPS effects, eritoran was administered i.v. (4 mg/kg body weight). Physical activity, peripheral blood pressure, and heart frequency were recorded before and after LPS and eritoran injection. In addition, intracardiac hemodynamic parameters were analyzed with a pressure conductance catheter. After 2 and 6 h of LPS stimulation ± eritoran treatment, the hearts and aortae were harvested, and TLR as well as inflammatory mediator expression was measured using reverse transcription–quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Lipopolysaccharide significantly decreased arterial blood pressure over time. Administration of eritoran partially prevented the LPS-dependent reduction in blood pressure and preserved cardiac function. In addition, LPS increased the expression of CD14 and TLR2 in cardiac and aortic tissue. In aortic tissue, eritoran attenuated this increase, whereas no significant reduction was observed in the heart. Furthermore, cardiac and aortic inducible nitric oxide synthetase mRNA levels were significantly increased 6 h after LPS application. This effect was reduced in the presence of eritoran. In summary, the beneficial influence of eritoran on cardiovascular function in vivo seems to rely mainly on reduction of LPS-induced inducible nitric oxide synthetase expression as well as on attenuated cytokine expression in the vascular wall.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses

There is interest in understanding posttranslational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down‐regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia‐inducible factor (HIF; via Cullin‐2). Here, we elucidate the role of human deneddylase‐1 (DEN‐1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN‐1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF‐1a, activated HIF promoter activity by 2.5‐fold, and induced HIF‐target genes in human epithelial cells up to 5‐fold. Knockdown of DEN‐1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5‐fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN‐1 is a regulator of cullin neddylation and fine‐tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.—Curtis, V. F., Ehrentraut, S. F., Campbell, E. L., Glover, L. E., Bayless, A., Kelly, C. J., Kominsky, D. J., Colgan, S. P., Stabilization of HIF through inhibition of Cullin‐2 neddylation is protective in mucosal inflammatory responses. FASEB J. 29, 208–215 (2015). www.fasebj.org

Central Role for Endothelial Human Deneddylase-1/SENP8 in Fine-Tuning the Vascular Inflammatory Response

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-α by assessing Cul neddylation status, NF-κB and HIF-1α stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α, and increased NF-κB/HIF-α promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity, and reduced secretion of TNF-α–elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.

Alpha-1-antitrypsin therapy ameliorates acute colitis and chronic murine ileitis

Background:Fecal alpha-1-antitrypsin (AAT) clearance has been a marker of clinical disease severity in inflammatory bowel diseases (IBDs) for many years. Although AAT deficiency is more often associated with lung and liver pathologies, AAT-deficient patients with concomitant IBD have been shown to develop more aggressive disease and rapid progression to surgery. Although recent studies have highlighted the pleiotropic anti-inflammatory functions of AAT, including reducing proinflammatory cytokine production and suppressing immune cell activation, its potential therapeutic role in IBD has not been described. Methods:The therapeutic potential of human AAT administration was assessed in murine models of IBD including new-onset and established chemically induced colitis and spontaneous chronic murine ileitis. Histological assessment of inflammation, cytokine secretion profiling, and flow cytometric evaluation of inflammatory infiltrate were performed in each model. The effect of AAT on intestinal barrier function was also examined both in vitro and in vivo. Results:AAT attenuated inflammation in small and large intestinal IBD models through reduced secretion of proinflammatory cytokines, inflammatory cell infiltration, and reduced tissue injury. AAT also increased intestinal restitution after chemically induced colitis. AAT significantly decreased intestinal permeability in vitro and in vivo as part of a protective mechanism for both acute and chronic models of IBD. Conclusions:Our findings describe a beneficial role for AAT in IBD models through suppression of cytokine production and enhanced intestinal barrier function. This raises the possibility that AAT supplementation, which has a long history of proven safety, may have a therapeutic effect in human IBD.

Adenosine and gastrointestinal inflammation

Nucleosides such as adenosine (Ado) influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides liberated at local sites of inflammation are metabolized through regulated phosphohydrolysis by a series of ecto-nucleotidases including ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73), found on the surface of a variety of cell types. Once generated, Ado is made available to bind and activate one of four G protein-coupled Ado receptors. Recent in vitro and in vivo studies implicate Ado in a broad array of tissue-protective mechanisms that provide new insight into adenosine actions. Studies in cultured cells and murine tissues have indicated that Ado receptors couple to novel posttranslational protein modifications, including Cullin deneddylation, as a new anti-inflammatory mechanism. Studies in Ado receptor-null mice have been revealing and indicate a particularly important role for the Ado A2B receptor in animal models of intestinal inflammation. Here, we review contributions of Ado to cell and tissue stress responses, with a particular emphasis on the gastrointestinal mucosa.