A Functional Network Driven by MicroRNA-125a Regulates Monocyte Trafficking in Acute Inflammation.

During the onset of acute inflammation, rapid trafficking of leukocytes is essential to mount appropriate immune responses towards an inflammatory insult. Monocytes are especially indispensable for counteracting the inflammatory stimulus, neutralising the noxa and reconstituting tissue homeostasis. Thus, monocyte trafficking to the inflammatory sites needs to be precisely orchestrated. In this study, we identify a regulatory network driven by miR-125a that affects monocyte adhesion and chemotaxis by the direct targeting of two adhesion molecules, i.e., junction adhesion molecule A (JAM-A), junction adhesion molecule-like (JAM-L) and the chemotaxis-mediating chemokine receptor CCR2. By investigating monocytes isolated from patients undergoing cardiac surgery, we found that acute yet sterile inflammation reduces miR-125a levels, concomitantly enhancing the expression of JAM-A, JAM-L and CCR2. In contrast, TLR-4-specific stimulation with the pathogen-associated molecular pattern (PAMP) LPS, usually present within the perivascular inflamed area, resulted in dramatically induced levels of miR-125a with concomitant repression of JAM-A, JAM-L and CCR2 as early as 3.5 h. Our study identifies miR-125a as an important regulator of monocyte trafficking and shows that the phenotype of human monocytes is strongly influenced by this miRNA, depending on the type of inflammatory stimulus.

Hypoxic-inflammatory responses under acute hypoxia: In Vitro experiments and prospective observational expedition trial.

Induction of hypoxia-inducible-factor-1α (HIF-1α) pathway and HIF-target genes allow adaptation to hypoxia and are associated with reduced incidence of acute mountain sickness (AMS). Little is known about HIF-pathways in conjunction with inflammation or exercise stimuli under acute hypobaric hypoxia in non-acclimatized individuals. We therefore tested the hypotheses that 1) both hypoxic and inflammatory stimuli induce hypoxic-inflammatory signaling pathways in vitro, 2) similar results are seen in vivo under hypobaric hypoxia, and 3) induction of HIF-dependent genes is associated with AMS in 11 volunteers. In vitro, peripheral blood mononuclear cells (PBMCs) were incubated under hypoxic (10%/5% O2) or inflammatory (CD3/CD28) conditions. In vivo, Interleukin 1ß (IL-1ß), C-X-C Chemokine receptor type 4 (CXCR-4), and C-C Chemokine receptor type 2 (CCR-2) mRNA expression, cytokines and receptors were analyzed under normoxia (520 m above sea level (a.s.l.)), hypobaric hypoxia (3883 m a.s.l.) before/after exercise, and after 24 h under hypobaric hypoxia. In vitro, isolated hypoxic (p = 0.004) or inflammatory (p = 0.006) stimuli induced IL-1ß mRNA expression. CCR-2 mRNA expression increased under hypoxia (p = 0.005); CXCR-4 mRNA expression remained unchanged. In vivo, cytokines, receptors, and IL-1ß, CCR-2 and CXCR-4 mRNA expression increased under hypobaric hypoxia after 24 h (all p ≤ 0.05). Of note, proinflammatory IL-1ß and CXCR-4 mRNA expression changes were associated with symptoms of AMS. Thus, hypoxic-inflammatory pathways are differentially regulated, as combined hypoxic and exercise stimulus was stronger in vivo than isolated hypoxic or inflammatory stimulation in vitro.

Myeloid-Derived Suppressor Cells Mediate Immunosuppression After Cardiopulmonary Bypass.

OBJECTIVES: Cardiopulmonary bypass is associated with severe immune dysfunctions. Particularly, a cardiopulmonary bypass-related long-lasting immunosuppressive state predisposes patients to a higher risk of postoperative complications, such as persistent bacterial infections. This study was conducted to elucidate mechanisms of post-cardiopulmonary bypass immunosuppression. DESIGN: In vitro studies with human peripheral blood mononuclear cells. SETTING: Cardiosurgical ICU, University Research Laboratory. PATIENTS: Seventy-one patients undergoing cardiac surgery with cardiopulmonary bypass (enrolled May 2017 to August 2018). INTERVENTIONS: Peripheral blood mononuclear cells before and after cardiopulmonary bypass were analyzed for the expression of immunomodulatory cell markers by real-time quantitative reverse transcription polymerase chain reaction. T cell effector functions were determined by enzyme-linked immunosorbent assay, carboxyfluorescein succinimidyl ester staining, and cytotoxicity assays. Expression of cell surface markers was assessed by flow cytometry. CD15 cells were depleted by microbead separation. Serum arginine was measured by mass spectrometry. Patient peripheral blood mononuclear cells were incubated in different arginine concentrations, and T cell functions were tested. MEASUREMENTS AND MAIN RESULTS: After cardiopulmonary bypass, peripheral blood mononuclear cells exhibited significantly reduced levels of costimulatory receptors (inducible T-cell costimulator, interleukin 7 receptor), whereas inhibitory receptors (programmed cell death protein 1 and programmed cell death 1 ligand 1) were induced. T cell effector functions (interferon γ secretion, proliferation, and CD8-specific cell lysis) were markedly repressed. In 66 of 71 patients, a not yet described cell population was found, which could be characterized as myeloid-derived suppressor cells. Myeloid-derived suppressor cells are known to impair immune cell functions by expression of the arginine-degrading enzyme arginase-1. Accordingly, we found dramatically increased arginase-1 levels in post-cardiopulmonary bypass peripheral blood mononuclear cells, whereas serum arginine levels were significantly reduced. Depletion of myeloid-derived suppressor cells from post-cardiopulmonary bypass peripheral blood mononuclear cells remarkably improved T cell effector function in vitro. Additionally, in vitro supplementation of arginine enhanced T cell immunocompetence. CONCLUSIONS: Cardiopulmonary bypass strongly impairs the adaptive immune system by triggering the accumulation of myeloid-derived suppressor cells. These myeloid-derived suppressor cells induce an immunosuppressive T cell phenotype by increasing serum arginine breakdown. Supplementation with L-arginine may be an effective measure to counteract the onset of immunoparalysis in the setting of cardiopulmonary bypass.

MicroRNAs as Clinical Biomarkers and Therapeutic Tools in Perioperative Medicine.

Over the past decade, evolutionarily conserved, noncoding small RNAs-so-called microRNAs (miRNAs)-have emerged as important regulators of virtually all cellular processes. miRNAs influence gene expression by binding to the 3'-untranslated region of protein-coding RNA, leading to its degradation and translational repression. In medicine, miRNAs have been revealed as novel, highly promising biomarkers and as attractive tools and targets for novel therapeutic approaches. miRNAs are currently entering the field of perioperative medicine, and they may open up new perspectives in anesthesia, critical care, and pain medicine. In this review, we provide an overview of the biology of miRNAs and their potential role in human disease. We highlight current paradigms of miRNA-mediated effects in perioperative medicine and provide a survey of miRNA biomarkers in the field known so far. Finally, we provide a perspective on miRNA-based therapeutic opportunities and perspectives.

Intronic miRNA-641 controls its host Gene's pathway PI3K/AKT and this relationship is dysfunctional in glioblastoma multiforme.

MicroRNAs have established their role as important regulators of the epigenome. A considerable number of human miRNA genes are found in intronic regions of protein-coding host genes, in many cases adopting their regulatory circuitry. However, emerging evidence foreshadows an unprecedented importance for this relationship: Intronic miRNAs may protect the cell from overactivation of the respective host pathway, a setting that may trigger tumor development. AKT2 is a well-known proto-oncogene central to the PI3K/AKT pathway. This pathway is known to promote tumor growth and survival, especially in glioblastoma. Its intronic miRNA, hsa-miR-641, is scarcely investigated, however. We hypothesized that miR-641 regulates its host AKT2 and that this regulation may become dysfunctional in glioblastoma. We found that indeed miR-641 expression differs significantly between GBM tissue and normal brain samples, and that transfection of glioma cells with miR-641 antagonizes the PI3K/AKT pathway. Combining clinical samples, cell cultures, and biomolecular methods, we could show that miR-641 doesn't affect AKT2's expression levels, but down-regulates kinases that are necessary for AKT2-activation, thereby affecting its functional state. We also identified NFAT5 as a miR-641 regulated central factor to trigger the expression of these kinases and subsequently activate AKT2. In summary, our study is the first that draws a connecting line between the proto-oncogene AKT2 and its intronic miRNA miR-641 with implication for glioblastoma development.

MicroRNA-146a controls Th1-cell differentiation of human CD4+ T lymphocytes by targeting PRKCε.

T-cell functions must be tightly controlled to keep the balance between vital proinflammatory activity and detrimental overactivation. MicroRNA-146a (miR-146a) has been identified as a key negative regulator of T-cell responses in mice. Its role in human T cells and its relevance to human inflammatory disease, however, remains poorly defined. In this study, we have characterized miR-146a-driven pathways in primary human T cells. Our results identify miR-146a as a critical gatekeeper of Th1-cell differentiation processes acting via molecular mechanisms not uncovered so far. MiR-146a targets protein kinase C epsilon (PRKCε), which is part of a functional complex consisting of PRKCε and signal transducer and activator of transcription 4 (STAT4). Within this complex, PRKCε phosphorylates STAT4, which in turn is capable of promoting Th1-cell differentiation processes in human CD4(+) T lymphocytes. In addition, we observed that T cells of sepsis patients had reduced levels of miR-146a and an increased PRKCε expression in the initial hyperinflammatory phase of the disease. Collectively, our results identify miR-146a as a potent inhibitor of Th1-cell differentiation in human T cells and suggest that dysregulation of miR-146a contributes to the pathogenesis of sepsis.

miR-124a and miR-155 enhance differentiation of regulatory T cells in patients with neuropathic pain.

BACKGROUND: Accumulating evidence indicates that neuropathic pain is a neuro-immune disorder with enhanced activation of the immune system. Recent data provided proof that neuropathic pain patients exhibit increased numbers of immunosuppressive regulatory T cells (Tregs), which may represent an endogenous attempt to limit inflammation and to reduce pain levels. We here investigate the molecular mechanisms underlying these alterations. METHODS: Our experimental approach includes functional analyses of primary human T cells, 3'-UTR reporter assays, and expression analyses of neuropathic pain patients' samples. RESULTS: We demonstrate that microRNAs (miRNAs) are involved in the differentiation of Tregs in neuropathic pain. We identify miR-124a and miR-155 as direct repressors of the histone deacetylase sirtuin1 (SIRT1) in primary human CD4(+) cells. Targeting of SIRT1 by either specific siRNA or by these two miRNAs results in an increase of Foxp3 expression and, consecutively, of anti-inflammatory Tregs (siRNA: 1.7 ± 0.4; miR-124a: 1.5 ± 0.4; miR-155: 1.6 ± 0.4; p < 0.01). As compared to healthy volunteers, neuropathic pain patients exhibited an increased expression of miR-124a (2.5 ± 0.7, p < 0.05) and miR-155 (1.3 ± 0.3; p < 0.05) as well as a reduced expression of SIRT1 (0.5 ± 0.2; p < 0.01). Moreover, the expression of these two miRNAs was inversely correlated with SIRT1 transcript levels. CONCLUSIONS: Our findings suggest that in neuropathic pain, enhanced targeting of SIRT1 by miR-124a and miR-155 induces a bias of CD4(+) T cell differentiation towards Tregs, thereby limiting pain-evoking inflammation. Deciphering miRNA-target interactions that influence inflammatory pathways in neuropathic pain may contribute to the discovery of new roads towards pain amelioration. TRIAL REGISTRATION: German Clinical Trial Register DRKS00005954.

Differential expression of P2X7 receptor and IL-1ß in nociceptive and neuropathic pain.

BACKGROUND: Despite substantial progress, pathogenesis and therapy of chronic pain are still the focus of many investigations. The ATP-gated P2X7 receptor (P2X7R) has previously been shown to play a central role in animal models of nociceptive inflammatory and neuropathic pain. Recently, we found that the adaptive immune system is involved in the pathophysiology of chronic nociceptive and neuropathic pain in humans. So far, data regarding P2X7R expression patterns on cells of the adaptive immune system of pain patients are scarce. We therefore analyzed the P2X7R expression on peripheral blood lymphocytes and monocytes, as well as serum levels of IL-1ß in patients suffering from chronic nociceptive and neuropathic pain in comparison to healthy volunteers in order to identify individuals who might benefit from a P2X7R modulating therapy. METHODS: P2X7R messenger RNA (mRNA) and protein expression were determined in patients with either chronic nociceptive low back pain (CLBP) or neuropathic pain (NeP), and in healthy volunteers by quantitative real-time PCR (qPCR) and by fluorescence-assisted cell-sorting (FACS), respectively. IL-1ß serum levels were measured with a multiplex cytokine assay. RESULTS: Compared to healthy volunteers, P2X7R mRNA (1.6-fold, p = 0.038) and protein levels were significantly increased on monocytes (NeP: 24.6 ± 6.2, healthy volunteers: 17.0 ± 5.4; p = 0.002) and lymphocytes (NeP: 21.8 ± 6.5, healthy volunteers: 15.6 ± 5.2; p = 0.009) of patients with NeP, but not in patients with CLBP. Similarly, IL-1ß serum concentrations were significantly elevated only in NeP patients (1.4-fold, p = 0.04). CONCLUSIONS: A significant upregulation of P2X7R and increased IL-1ß release seems to be a particular phenomenon in patients with NeP. P2X7R inhibitors may therefore represent a potential option for the treatment of this frequently intractable type of pain. German Clinical Trial Register (DRKS): Registration Trial DRKS00005954.

Emerging Roles for MicroRNAs in Perioperative Medicine.

MicroRNAs (miRNAs) are small, non-protein-coding, single-stranded RNAs. They function as posttranscriptional regulators of gene expression by interacting with target mRNAs. This process prevents translation of target mRNAs into a functional protein. miRNAs are considered to be functionally involved in virtually all physiologic processes, including differentiation and proliferation, metabolism, hemostasis, apoptosis, and inflammation. Many of these functions have important implications for anesthesiology and critical care medicine. Studies indicate that miRNA expression levels can be used to predict the risk for eminent organ injury or sepsis. Pharmacologic approaches targeting miRNAs for the treatment of human diseases are currently being tested in clinical trials. The present review highlights the important biological functions of miRNAs and their usefulness as perioperative biomarkers and discusses the pharmacologic approaches that modulate miRNA functions for disease treatment. In addition, the authors discuss the pharmacologic interactions of miRNAs with currently used anesthetics and their potential to impact anesthetic toxicity and side effects.

Down-regulation of MicroRNA-31 in CD4+ T Cells Contributes to Immunosuppression in Human Sepsis by Promoting TH2 Skewing.

BACKGROUND: Immunosuppression has been recognized as a major cause of sepsis-related mortality. Currently, there is much interest in identifying central hubs controlling septic immunoparalysis. In this context, in this study, the authors investigate the role of microRNA-31 (miR-31) as a regulator of T cell functions. METHODS: Primary human T cells were separated from healthy volunteers (n = 16) and from sepsis patients by magnetic beads (n = 23). Expression of mRNA/microRNA (miRNA) was determined by real-time polymerase chain reaction. Gene silencing was performed by small interfering RNA transfection, and miRNA-binding sites were validated by reporter gene assays. Effects of miR-31 or anti-miR-31 transfection were analyzed by real-time polymerase chain reaction, Western blotting, and flow cytometry. RESULTS: Overexpression of miR-31 in stimulated CD4 T cells promoted a proinflammatory phenotype with increased levels of interferon-γ (1.63 ± 0.43; P = 0.001; means ± SD) and reduced expression of interleukin (IL)-2 (0.66 ± 0.19; P = 0.005) and IL-4 (0.80 ± 0.2; P = 0.0001). In contrast, transfection of anti-miR-31 directed cells toward a TH2 phenotype. Effects on IL-2 and IL-4 were mediated by targeting of nuclear factor-kappa B-inducing kinase and factor-inhibiting hypoxia-inducible factor-1α. Interferon-γ, however, was influenced via control of signaling lymphocytic activation molecule (SLAM)-associated protein, an essential adaptor molecule of immunomodulatory SLAM receptor signaling, which was identified as a novel target gene of miR-31. In sepsis patients, an epigenetically driven down-regulation of miR-31 was found (0.44 ± 0.25; P = 0.0001), associated with increased nuclear factor-kappa B-inducing kinase, factor-inhibiting hypoxia-inducible factor-1α, SLAM-associated protein expression, and a cytokine shift toward TH2. CONCLUSIONS: In this study, the authors provide novel evidence of miR-31 as an emerging key posttranscriptional regulator of sepsis-associated immunosuppression. The study results contribute to a further understanding of septic immunoparalysis and provide new perspectives on miRNA-based diagnostic approaches.

Dynamic 18F-FET PET in suspected WHO grade II gliomas defines distinct biological subgroups with different clinical courses.

In suspected grade II gliomas, three distinct patterns of time-activity curves (TAC) on O-(2-[(18)F]fluoroethyl)-1-tyrosine ((18)F-FET) positron emission tomography (PET) have been delineated (i) increasing TAC homogeneously throughout the tumor, and decreasing TAC, (ii) either homogeneously throughout the tumor or (iii) only focally within otherwise increasing TAC patterns. Increasing TAC was associated with low-grade histology and decreasing TAC with high-grade histology. This prospective study analyzed whether these patterns correlate with distinct biological tumor subtypes and differential outcome. (18)F-FET PET-guided biopsies were used for stepwise histopathological evaluation. Molecular-genetic evaluation included O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation, isocitrate dehydrogenase (IDH1/2) mutational and 1p/19q codeletion status. Progression-free survival (PFS) was estimated with the Kaplan-Meier method. Prognostic factors were obtained from multivariate regression models. 98 adult patients were included. Homogeneous increasing, focal decreasing and homogeneous decreasing TAC were seen in 51, 19 and 28 patients. The corresponding 1-year (2-years) PFS were 92% (85%), 89% (51%) and 50% (28%; p = 0.002). IDH1/2 mutations were more frequent in tumors with homogeneous increasing (90%) and focal decreasing (79%) TAC, but were rare in those exhibiting homogeneous decreasing TAC (25%; p < 0.001). Overall, TAC patterns, IDH1/2 mutational and 1p/19q codeletion status were powerful and independent prognostic factors. Dynamic (18)F-FET PET might be an important and independent imaging biomarker for patients with suspected WHO grade II gliomas and offers perspectives for stratified diagnostic and therapeutic strategies. Tumors with focal decreasing TAC need highly targeted surgical interventions to avoid undergrading and undertreatment.

Anti-inflammatory T-cell shift in neuropathic pain.

BACKGROUND: The classification of pain into nociceptive and neuropathic pain is based on characteristic symptoms and different pathophysiological mechanisms. In a recent investigation, we found a disrupted TH17/Treg balance in patients suffering from chronic unspecific low back pain (CLBP). These patients did not show any signs of neuropathy. There is evidence for a considerable impact of the immune system also in neuropathic pain. However, the role of the adaptive immune system is still unclear. In the present study, we investigated systemic T-cell subset responses and T-cell related cytokine profiles in patients with chronic neuropathic pain. METHODS: We analyzed T-cell subsets, mRNA expression and T-cell-related cytokine profiles in 26 patients suffering from neuropathic pain in comparison to 26 healthy controls. Using multicolor flow cytometry (FACS), we quantified the number of T helper cells 1 (TH1), TH2, TH17 and regulatory T-cells (Tregs). Forkhead-Box-Protein 3 (FoxP3), Transforming growth factor-ß (TGF-ß) and RAR-related orphan receptor-γT (ROR-γT) mRNA expression was determined by quantitative real-time PCR (qPCR) and levels of pain-related cytokines were measured by Human Cytokine Multiplex Immunoassay (Macrophage inflammatory protein-1α (MIP-1α), Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), Interleukin (IL) -4, IL-6, IL-10, IL-17, and IL-23). RESULTS: We found a TH17/Treg imbalance with significantly increased anti-inflammatory Tregs and decreased pro-inflammatory TH17 cells in patients with neuropathic pain as compared to healthy controls. These results were confirmed on mRNA level: Treg-related FoxP3 and TGF-ß mRNA expression was elevated, whereas expression of TH17-related RORγT was reduced. Cytokine analyses revealed only marginal changes. CONCLUSIONS: Our investigation revealed a clear shift of T-cell subsets towards anti-inflammation in patients with neuropathic pain. Interestingly, this is quite similar to our previous findings in CLBP patients, but even more pronounced. Therefore, it remains to be elucidated in future investigations whether the immune changes represent an underlying pathophysiological mechanism or an epiphenomenon induced by ongoing pain and stress. GERMAN CLINICAL TRIAL REGISTER (DRKS): Trial registration number: DRKS00005954.

MicroRNA-665 is involved in the regulation of the expression of the cardioprotective cannabinoid receptor CB2 in patients with severe heart failure.

The myocardial endocannabinoid system has been linked to stress response and cardioprotection. In chronic heart failure (CHF), protective CB2 receptors are markedly up-regulated while CB1 receptors are slightly down-regulated. We here provide evidence that myocardial CB receptors are subject to microRNA regulation. By a combined computational and experimental approach we show that CB1 receptors are regulated by miR-494, and CB2 receptors are targeted by miR-665. Moreover, we demonstrate that in CHF, miR-665 expression is significantly decreased while miR-494 is slightly increased, which is concordant with the previously reported alterations of CB receptors. These results suggest that in CHF, altered expression of specific miRNAs may contribute to a compensatory response of the diseased myocardium.

In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing.

Favorable outcome after chemotherapy of glioblastomas cannot unequivocally be linked to promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene encoding a DNA repair enzyme associated with resistance to alkylating agents. This indicates that molecular mechanisms determining MGMT expression have not yet been fully elucidated. We here show that glioblastomas are capable to downregulate MGMT expression independently of promoter methylation by elongation of the 3'-UTR of the mRNA, rendering the alternatively polyadenylated transcript susceptible to miRNA-mediated suppression. While the elongated transcript is poorly expressed in normal brain, its abundance in human glioblastoma specimens is inversely correlated with MGMT mRNA expression. Using a bioinformatically guided experimental approach, we identified miR-181d, miR-767-3p, and miR-648 as significant post-transcriptional regulators of MGMT in glioblastomas; the first two miRNAs induce MGMT mRNA degradation, the latter affects MGMT protein translation. A regression model including the two miRNAs influencing MGMT mRNA expression and the MGMT methylation status reliably predicts The Cancer Genome Atlas MGMT expression data. Responsivity of MGMT expressing T98G glioma cells to temozolomide was significantly enhanced after transfection of miR-181d, miR-767-3p, and miR-648. Taken together, our results uncovered alternative polyadenylation of the MGMT 3'-UTR and miRNA targeting as new mechanisms of MGMT silencing.

[Immunomodulation by nutritional intervention in critically ill patients]. / Immunmodulation durch Ernährung bei kritisch kranken Patienten.

Critically ill patients often suffer from a complex and severe immunological dysfunction. The differentiation and function of human immune cells are fundamentally controlled through metabolic processes. New concepts of immunonutrition therefore try to use enteral and parenteral nutrition to positively impact on the immune function of intensive care unit patients. This review article concisely presents the currently available evidence on the commonly used isolated supplements (anti-oxidative substances, amino acids, essential fatty acids) and difficulties related to their clinical use. The second part presents new and more comprehensive concepts of immunonutrition to influence the intestinal microbiome and to modulate the macronutrient composition. Immunonutrition of critically ill patients bears enormous potential and could become a valuable clinical tool for modulation of the immunometabolism of intensive care unit patients.

A ketogenic diet substantially reshapes the human metabolome.

BACKGROUND: Western dietary habits (WD) have been shown to promote chronic inflammation, which favors the development of many of today's non-communicable diseases. Recently, ketogenic diets (KD) have emerged as an immune-regulating countermeasure for WD-induced metaflammation. To date, beneficial effects of KD have been solely attributed to the production and metabolism of ketone bodies. Given the drastic change in nutrient composition during KD, it is reasonable to assume that there are widespread changes in the human metabolome also contributing to the impact of KD on human immunity. The current study was conducted to gain insight into the changes of the human metabolic fingerprint associated with KD. This could allow to identify metabolites that may contribute to the overall positive effects on human immunity, but also help to recognize potential health risks of KD. METHODS: We conducted a prospective nutritional intervention study enrolling 40 healthy volunteers to perform a three-week ad-libitum KD. Prior to the start and at the end of the nutritional intervention serum metabolites were quantified, untargeted mass spectrometric metabolome analyses and urine analyses of the tryptophan pathway were performed. RESULTS: KD led to a marked reduction of insulin (-21.45% ± 6.44%, p = 0.0038) and c-peptide levels (-19.29% ± 5.45%, p = 0.0002) without compromising fasting blood glucose. Serum triglyceride concentration decreased accordingly (-13.67% ± 5.77%, p = 0.0247), whereas cholesterol parameters remained unchanged. LC-MS/MS-based untargeted metabolomic analyses revealed a profound shift of the human metabolism towards mitochondrial fatty acid oxidation, comprising highly elevated levels of free fatty acids and acylcarnitines. The serum amino acid (AA) composition was rearranged with lower abundance of glucogenic AA and an increase of BCAA. Furthermore, an increase of anti-inflammatory fatty acids eicosatetraenoic acid (p < 0.0001) and docosahexaenoic acid (p = 0.0002) was detected. Urine analyses confirmed higher utilization of carnitines, indicated by lower carnitine excretion (-62.61% ± 18.11%, p = 0.0047) and revealed changes to the tryptophan pathway depicting reduced quinolinic acid (-13.46% ± 6.12%, p = 0.0478) and elevated kynurenic acid concentrations (+10.70% ± 4.25%, p = 0.0269). CONCLUSIONS: A KD fundamentally changes the human metabolome even after a short period of only three weeks. Besides a rapid metabolic switch to ketone body production and utilization, improved insulin and triglyceride levels and an increase in metabolites that mediate anti-inflammation and mitochondrial protection occurred. Importantly, no metabolic risk factors were identified. Thus, a ketogenic diet could be considered as a safe preventive and therapeutic immunometabolic tool in modern medicine. TRIAL REGISTRATION: German Clinical Trials Register; DRKS-ID: DRKS00027992 (www.drks.de).

IDH1 mutations in grade II astrocytomas are associated with unfavorable progression-free survival and prolonged postrecurrence survival.

BACKGROUND: The favorable prognostic impact of mutations in the IDH1 gene is well documented for malignant gliomas; its influence on World Health Organization (WHO) grade II astrocytomas, however, is still under debate. METHODS: A previously published database of 127 predominantly surgically treated patients harboring WHO grade II astrocytomas was revisited. Patients were screened for TP53 mutations (sequencing analysis), IDH1 mutations (pyrosequencing), and MGMT promoter methylation (methylation-specific polymerase chain reaction and bisulfite sequencing). Endpoints were overall survival, progression-free survival (PFS), time to malignant transformation, and postrecurrence survival. Radiotherapy was usually withheld until tumor progression/malignant transformation occurred. RESULTS: IDH1 mutations, TP53 mutations, and methylated MGMT promoters were seen in 78.1%, 51.2%, and 80.0% of the analyzed tumors, respectively. IDH1 mutations, which were significantly associated with TP53 mutations and/or MGMT promoter methylation (P < .001), resulted in shortened PFS (median, 47 vs 84 months; P = .004); postrecurrence survival, however, was significantly increased in those patients undergoing malignant transformation (median, 49 vs 13.5 months; P = .006). Overall survival was not affected by IDH1. A similar pattern of influence was seen for MGMT promoter methylation. Methylated tumors did significantly worse (better) in terms of PFS (postrecurrence survival); a low number of unmethylated tumors, however, limited the power of this analysis. Conversely, TP53 mutations were stringently associated with a worse prognosis throughout the course of the disease. CONCLUSIONS: IDH1 mutations are associated with a Janus headlike phenomenon; unfavorable prognostic influence on PFS turns into favorable impact on postrecurrence survival. A similar pattern of influence might exist for MGMT methylation.

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-α (mediating anti-inflammatory effects) and glucocorticoid receptor-ß (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-α 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-ß by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-α-specific 3'-untranslated-region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-α 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-α, glucocorticoid receptor-ß, 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-α (not glucocorticoid receptor-ß) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-ß doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-α. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-α and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-ß 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-α thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-ß levels.

Ketone Bodies Improve Human CD8+ Cytotoxic T-Cell Immune Response During COVID-19 Infection.

Severe COVID-19 is characterized by profound CD8+ T-cell dysfunction, which cannot be specifically treated to date. We here investigate whether metabolic CD8+ T-cell reprogramming by ketone bodies could be a promising strategy to overcome the immunoparalysis in COVID-19 patients. This approach was triggered by our recent pioneering study, which has provided evidence that CD8+ T-cell capacity in healthy subjects could be significantly empowered by a Ketogenic Diet. These improvements were achieved by immunometabolic rewiring toward oxidative phosphorylation. We here report similar strengthening of CD8+ T cells obtained from severely diseased COVID-19 patients: Flow cytometry and ELISA revealed elevated cytokine expression and secretion (up to + 24%) upon ketone treatment and enhanced cell lysis capacity (+ 21%). Metabolic analyses using Seahorse technology revealed upregulated mitochondrial respiratory chain activity (+ 25%), enabling both superior energy supply (+ 44%) and higher mitochondrial reactive oxygen species signaling. These beneficial effects of ketones might represent evolutionary conserved mechanisms to strengthen human immunity. Our findings pave the road for metabolic treatment studies in COVID-19.

Cell-Crossing Functional Network Driven by microRNA-125a Regulates Endothelial Permeability and Monocyte Trafficking in Acute Inflammation.

Opening of the endothelial barrier and targeted infiltration of leukocytes into the affected tissue are hallmarks of the inflammatory response. The molecular mechanisms regulating these processes are still widely elusive. In this study, we elucidate a novel regulatory network, in which miR-125a acts as a central hub that regulates and synchronizes both endothelial barrier permeability and monocyte migration. We found that inflammatory stimulation of endothelial cells induces miR-125a expression, which consecutively inhibits a regulatory network consisting of the two adhesion molecules VE-Cadherin (CDH5) and Claudin-5 (CLDN5), two regulatory tyrosine phosphatases (PTPN1, PPP1CA) and the transcription factor ETS1 eventually leading to the opening of the endothelial barrier. Moreover, under the influence of miR-125a, endothelial expression of the chemokine CCL2, the most predominant ligand for the monocytic chemokine receptor CCR2, was strongly enhanced. In monocytes, on the other hand, we detected markedly repressed expression levels of miR-125a upon inflammatory stimulation. This induced a forced expression of its direct target gene CCR2, entailing a strongly enhanced monocyte chemotaxis. Collectively, cell-type-specific differential expression of miR-125a forms a synergistic functional network controlling monocyte trafficking across the endothelial barrier towards the site of inflammation. In addition to the known mechanism of miRNAs being shuttled between cells via extracellular vesicles, our study uncovers a novel dimension of miRNA function: One miRNA, although disparately regulated in the cells involved, directs a biologic process in a synergistic and mutually reinforcing manner. These findings provide important new insights into the regulation of the inflammatory cascade and may be of great use for future clinical applications.