Carola Ledderose

Corticosteroid resistance in sepsis is influenced by microrna-124–induced downregulation of glucocorticoid receptor-α*

Objective: Acquired glucocorticoid resistance frequently complicates the therapy of sepsis. It leads to an exaggerated proinflammatory response and has been related to altered expression profiles of glucocorticoid receptor isoforms glucocorticoid receptor-&agr; (mediating anti-inflammatory effects) and glucocorticoid receptor-&bgr; (acting as a dominant negative inhibitor). We investigated the impact of glucocorticoid receptor isoforms on glucocorticoid effects in human T-cells. We hypothesized that 1) changes of the ratio of glucocorticoid receptor isoforms impact glucocorticoid resistance and 2) glucocorticoid receptor-&agr; expression is controlled by microRNA-mediated gene silencing. Design: Laboratory-based study. Setting: University research laboratory. Subjects and Patients: Healthy volunteers, sepsis patients. Methods: First, T-cells from healthy volunteers (native and CD3/CD28-stimulated cells with or without addition of hydrocortisone) were analyzed for the expression of glucocorticoid receptor-isoforms by quantitative polymerase chain reaction. Additionally, effects of gene silencing of glucocorticoid receptor-&bgr; by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-&agr;–specific 3′-untranslated-region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-&agr; expression were analyzed in Jurkat T-cells and in T-cells from healthy volunteers (quantitative polymerase chain reaction and Western blotting). Finally, expression of glucocorticoid receptor-&agr;, glucocorticoid receptor-&bgr;, and miR-124 was tested in T-cells of sepsis patients (n = 24). Measurements and Main Results: Stimulation of T-cells induced a significant upregulation of glucocorticoid receptor-&agr; (not glucocorticoid receptor-&bgr;) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-&bgr; doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-&agr;. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-&agr; and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-&bgr; expression was two-fold upregulated (p < .01) and exhibited a remarkable interindividual variability. Conclusions: Glucocorticoid treatment induces expression of miR-124, which downregulates glucocorticoid receptor-&agr; thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-&bgr; levels.

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.

CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

Pannexin 1 Channels Link Chemoattractant Receptor Signaling to Local Excitation and Global Inhibition Responses at the Front and Back of Polarized Neutrophils

Background: Chemotaxis requires excitatory and inhibitory signals at the front and back of cells. PANX1 and P2Y2 receptors provide local excitation at the front. Results: We found that PANX1 triggers A2A receptor activation and inhibits chemotactic signaling at the back. Conclusion: PANX1 thus integrates excitatory and inhibitory signals that regulate chemotaxis at the front and back of cells. Significance: These findings suggest new strategies to modulate neutrophil chemotaxis. Neutrophil chemotaxis requires excitatory signals at the front and inhibitory signals at the back of cells, which regulate cell migration in a chemotactic gradient field. We have previously shown that ATP release via pannexin 1 (PANX1) channels and autocrine stimulation of P2Y2 receptors contribute to the excitatory signals at the front. Here we show that PANX1 also contributes to the inhibitory signals at the back, namely by providing the ligand for A2A adenosine receptors. In resting neutrophils, we found that A2A receptors are uniformly distributed across the cell surface. In polarized cells, A2A receptors redistributed to the back where their stimulation triggered intracellular cAMP accumulation and protein kinase A (PKA) activation, which blocked chemoattractant receptor signaling. Inhibition of PANX1 blocked A2A receptor stimulation and cAMP accumulation in response to formyl peptide receptor stimulation. Treatments that blocked endogenous A2A receptor signaling impaired the polarization and migration of neutrophils in a chemotactic gradient field and resulted in enhanced ERK and p38 MAPK signaling in response to formyl peptide receptor stimulation. These findings suggest that chemoattractant receptors require PANX1 to trigger excitatory and inhibitory signals that synergize to fine-tune chemotactic responses at the front and back of neutrophils. PANX1 channels thus link local excitatory signals to the global inhibitory signals that orchestrate chemotaxis of neutrophils in gradient fields.

Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

BackgroundThe choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes.ResultsThe mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells.ConclusionsThe study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

Mitochondria Are Gate-keepers of T Cell Function by Producing the ATP That Drives Purinergic Signaling

Background: Autocrine purinergic signaling regulates T cell function. Results: We found that mitochondria are the source of ATP that drives these signaling mechanisms. Conclusion: Mitochondria are the gate-keepers of T cell function that control purinergic signaling and T cell activation. Significance: These findings suggest that mitochondria could be therapeutic targets to modulate T cell responses. T cells play a central role in host defense. ATP release and autocrine feedback via purinergic receptors has been shown to regulate T cell function. However, the sources of the ATP that drives this process are not known. We found that stimulation of T cells triggers a spike in cellular ATP production that doubles intracellular ATP levels in <30 s and causes prolonged ATP release into the extracellular space. Cell stimulation triggered rapid mitochondrial Ca2+ uptake, increased oxidative phosphorylation, a drop in mitochondrial membrane potential (Δψm), and the accumulation of active mitochondria at the immune synapse of stimulated T cells. Inhibition of mitochondria with CCCP, KCN, or rotenone blocked intracellular ATP production, ATP release, intracellular Ca2+ signaling, induction of the early activation marker CD69, and IL-2 transcription in response to cell stimulation. These findings demonstrate that rapid activation of mitochondrial ATP production fuels the purinergic signaling mechanisms that regulate T cells and define their role in host defense.

Altered myocardial expression of ghrelin and its receptor (GHSR-1a) in patients with severe heart failure

Ghrelin is a peptide hormone mainly produced by the stomach, which strongly stimulates the release of growth hormone (GH) via the GH secretagogue receptor 1a (GHSR-1a) located in the hypothalamus. It has been reported to exert performance-enhancing effects on myocardial function, and as both ghrelin and GHSR-1a are expressed in myocardial tissues, the ghrelin system may have a direct GH-independent impact on cardiac function. We intended to investigate the expression of ghrelin and its receptor GHSR-1a in different myocardial areas of patients with chronic heart failure (CHF) as compared to heart-healthy subjects to better define the role of the ghrelin signaling system in the networks regulating cardiac function and its potential as a target for diagnosis and/or treatment of CHF. Myocardium biopsies of 12 patients undergoing heart transplantation and suffering from CHF were obtained. Expression of both ghrelin and GHSR-1a was assessed by means of immunohistochemistry and real-time PCR. Expression of ghrelin was significantly decreased in CHF hearts both in atrium and ventricles in comparison to the control hearts (p<0.05). The expression of the GHS-1a receptor was significantly increased in the CHF biopsies as compared to controls (p<0.05). No significant differences were found between the anatomical areas studied. Expression of myocardial ghrelin and GHSR-1a is directly associated with myocardial function: CHF hearts exhibit an impaired ghrelin production which might reflect maladaptive processes and an - probably compensatory - increase in GHSR-1a expression. These findings may open up new perspectives regarding the potential of ghrelin signaling as a target for pharmacological modulation.

Mitochondria regulate neutrophil activation by generating ATP for autocrine purinergic signaling.

Background: Purinergic signaling regulates neutrophil activation. The source of ATP required for this process is unknown. Results: Mitochondria form the ATP that initiates purinergic signaling, and glycolysis provides the ATP that sustains functional responses of activated neutrophils. Conclusion: Mitochondrial ATP has a central role in triggering neutrophil activation. Significance: Mitochondria are regulators of the innate immune defense provided by neutrophils. Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y2 receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca2+ signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca2+ signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca2+ signaling via P2X receptor-mediated Ca2+ influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.

Ghrelin, A Novel Peptide Hormone in the Regulation of Energy Balance and Cardiovascular Function

Ghrelin, a peptide hormone predominantly produced by the stomach, is a potent stimulator of growth hormone release, food intake and weight gain. Besides its functions in regulating energy homeostasis, ghrelin has pronounced cardioprotective effects and was shown to improve cardiac performance in chronic heart failure (CHF). The multifunctional nature of ghrelin makes it an interesting pharmacological target for various diseases. Inhibition of ghrelin could be a promising approach in obesity-related disorders, while an enhancement of the ghrelin response is considered beneficial in several pathologic conditions marked by malnutrition, wasting and cachexia, including CHF, cancer, chronic pulmonary disease or chronic infections. In particular, patients suffering from CHF could possibly benefit from ghrelin based compounds that do not only help to reverse cardiac cachexia - by inducing a positive energy balance - but also enhance the direct cardioprotective effects of ghrelin. This review highlights the role of ghrelin in the regulation of energy balance and cardiovascular function and summarizes the most recent patents, developments and strategies in ghrelin-based pharmacotherapy for the treatment of pathologic conditions associated with obesity, cachexia or cardiovascular dysfunction.

IMMUNOMODULATORY PROPERTIES OF PENTOXIFYLLINE ARE MEDIATED VIA ADENOSINE-DEPENDENT PATHWAYS

The phosphodiesterase inhibitor pentoxifylline (PTX) exerts multiple beneficial immunomodulatory effects in states of hyperinflammation. However, the exact mechanism of action still remains elusive, and the clinical effects of PTX cannot be reliably predicted. In immune cells, the G protein-coupled adenosine A2A receptor (A2AR) exerts strong anti-inflammatory effects. As PTX amplifies signaling pathways downstream of Gs protein-coupled receptors, the A2AR-signaling pathway might be involved in the mediation of immune-suppressive effects of PTX. Here, we investigated this assumption in LPS-stimulated human polymorphonuclear (PMN) leukocytes and in anti-CD3/CD28-stimulated human T cells. In stimulated PMN leukocytes, PTX treatment led to a 4.5-fold decrease of the 50% inhibitory concentrations of adenosine on the H2O2 production; i.e., for adenosine plus PTX (in clinically relevant concentrations), an overadditive increase of inhibitory effects from less than 20% (estimated for each) to 56% (±5%) was found. In T cells, adenosine plus PTX revealed similar synergistic inhibitory effects on proinflammatory cytokine production. Inhibition of interferon &ggr; and TNF-&agr; production increased from 7% (±1%) and 31% (±6%) (PTX alone) to 49% (±2%) and 69% (±6%), respectively. In T cells and PMN leukocytes, mRNA transcription of the A2AR was significantly increased upon stimulation, which was not influenced by PTX. In human PMN leukocytes and T cells, clinically relevant anti-inflammatory effects of PTX can be achieved only in the presence of sufficient adenosine concentrations. Sufficient adenosine levels might be a prerequisite for the accessibility of sepsis patients to treatment with PTX.