Background/Objectives: The ketogenic diet (KD) has been shown to exert beneficial effects on human immunity by enhancing cytotoxic T lymphocyte function through metabolic reprogramming. However, strict dietary restrictions limit adherence and complicate its use in clinical practice. Exogenous ketone supplements have therefore been promoted as a more feasible alternative to elevate ketone body levels without the need for dietary changes. The objective of this study was to assess whether ketone salt or ketone ester supplementation can reproduce KD-mediated immunometabolic effects on CD8+ T cells in healthy individuals. Methods: In a prospective interventional study, healthy volunteers received either ketone salts (KS) or ketone esters (KE) for three weeks. Plasma β-hydroxybutyrate (BHB) concentrations were determined, and CD8+ T-cell cytokine secretion, functional responses, and mitochondrial energy metabolism were analyzed. In a subgroup, KS supplementation was combined with a carbohydrate-restricted, non-ketogenic diet. Results: While KS supplementation resulted in a short-lived increase in plasma BHB concentrations followed by increased BHB uptake in immune cells, KE supplementation led to more sustained plasma BHB levels, however, without detectable intracellular BHB accumulation. Neither intervention affected CD8+ T-cell cytokine production, functional capacity, or mitochondrial energy metabolism, and KS intake combined with a carbohydrate-restricted, non-ketogenic diet likewise did not alter CD8+ T-cell immunometabolic parameters. Conclusions: Transient elevation of circulating ketone body levels through supplementation seems insufficient to reproduce the immunometabolic effects of a KD, which likely require broader metabolic adaptations. Thus, the impact of exogenous ketones on adaptive immunity in healthy individuals appears limited.

BackgroundBrain and body metabolic disruptions have been suggested in several studies as a mechanism underlying posttraumatic stress disorder (PTSD). Therapies targeting metabolic pathways might provide new treatment options. This review explores the potential of ketogenic therapies, such as the ketogenic diet and ketone supplementation, as novel metabolic treatments for PTSD.MethodsAn integrative review was conducted. We searched PubMed, Scopus, PsycINFO, PsycArticles, PSYNDEX, MEDLINE, ClinicalTrials.gov, International Clinical Trials Registry Platform, and EU Clinical Trials Register for clinical, preclinical, observational, mechanistic, and review studies that reported outcomes or mechanisms on the intersection of ketogenic therapy and PTSD. Methodological quality of included studies was assessed independently by two reviewers based on predefined criteria.ResultsSixteen articles (9 human, 4 animal studies, 3 reviews) were eligible and included in the present review. Several metabolic and signaling pathways through which ketogenic therapies could impact PTSD pathophysiology were identified. Preclinical data suggestive of potential efficacy were identified, while only minimal reliable information is available from human studies.DiscussionImportantly, the absence of larger clinical trials and the reliance on low-level evidence substantially limit current interpretations. High-quality randomized and adequately powered clinical trials are required to examine the potential role of ketogenic therapies in treating PTSD.

Cytotoxic T cell (CTL) dysfunction is a hallmark of immune paralysis after major surgery, increasing susceptibility to severe nosocomial infections and contributing to mortality in critically ill patients. The mechanisms remain poorly understood. We demonstrate that reactive oxygen species (ROS) released by myeloid-derived suppressor cells (MDSC) transiently emerging after surgery, drive perioperative CTL immunoparalysis. These ROS damage CTL mitochondria, triggering secondary mitochondrial ROS amplification and overwhelming antioxidant defenses. The resulting oxidative cascade impairs oxidative phosphorylation and suppresses CTL effector function. Concurrently, stress-induced mitochondrial hyperfusion disrupts fission-dependent translocation to the immunological synapse, exacerbating bioenergetic failure. MitoTEMPO, a mitochondria-targeted antioxidant, partially mitigates these effects, highlighting mitochondrial stabilization as a potential strategy to prevent perioperative immune dysfunction.

Patients with sepsis experience metabolic and immunologic dysfunction that may be amplified by standard carbohydrate-based nutrition. A ketogenic diet (KD) may offer an immunologically advantageous alternative, although clinical evidence is limited. We conducted a single-center, open-label, randomized controlled trial to assess whether a KD could induce stable ketosis in critically ill patients with sepsis. Secondary outcomes included assessment of feasibility and safety of KD, as well as explorative analysis of clinical and immunological characteristics. Forty critically ill adults were randomized to either a ketogenic or standard high-carbohydrate diet. Stable ketosis was achieved in all KD patients, with significant increases in β-hydroxybutyrate levels compared with controls [mean difference 1.4 milimoles per liter; 95% confidence interval (CI): 1.0 to 1.8; P < 0.001). No major adverse events or harmful metabolic side effects (acidosis, dysglycemia, or dyslipidemia) were observed. After day 4, none of the patients in the KD group required insulin treatment, whereas in the control group, insulin dependency ranged between 35% and 60% ( P = 0.009). There were no differences in 30-day survival, but ventilation-free [incidence rate ratio (IRR) 1.7; 95% CI: 1.5 to 2.1; P < 0.001], vasopressor-free (IRR 1.7; 95% CI: 1.5 to 2.0; P < 0.001), dialysis-free (IRR 1.5; 95% CI: 1.3 to 1.8; P < 0.001), and intensive care unit–free days (IRR 1.7; 95% CI: 1.4 to 2.1; P < 0.001) were higher in the ketogenic group. Next-generation sequencing of CD4 + /CD8 + T cells and protein analyses showed reduced immune dysregulation, with decreased gene expression of T-cell activation and signaling markers and lower pro-inflammatory cytokine secretion. This trial demonstrated the safe induction of a stable ketogenic state in sepsis, warranting larger trials to investigate potential benefits in sepsis-related organ dysfunction.

A ketogenic diet (KD) is increasingly debated as a countermeasure against nutrition-related modern diseases. While being immunologically beneficial, KD is still suspected of having severe metabolic side effects and negatively impacting general well-being, which prevents its widespread clinical use. We conducted a prospective pre-post interventional study investigating the effects of an eucaloric KD on metabolism, weight loss, body composition, diet adherence, and quality of life. The study had two stages: first, feasibility was tested in healthy, normal-weight participants over three weeks. After positive results, the KD period was expanded to three months, enrolling adults with overweight. Significant weight loss was observed in both groups, reducing body fat without affecting muscle or bone mass and without adverse metabolic changes. Quality of life improved, and fatigue symptoms in subjects with overweight decreased. These findings may help to overcome reservations about KD, encouraging its use as a medical tool for treating nutrition-related disorders.

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

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).

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.

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.

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.

Very-low-carbohydrate diet triggers the endogenous production of ketone bodies as alternative energy substrates. There are as yet unproven assumptions that ketone bodies positively affect human immunity. We have investigated this topic in an in vitro model using primary human T cells and in an immuno-nutritional intervention study enrolling healthy volunteers. We show that ketone bodies profoundly impact human T-cell responses. CD4(+), CD8(+), and regulatory T-cell capacity were markedly enhanced, and T memory cell formation was augmented. RNAseq and functional metabolic analyses revealed a fundamental immunometabolic reprogramming in response to ketones favoring mitochondrial oxidative metabolism. This confers superior respiratory reserve, cellular energy supply, and reactive oxygen species signaling. Our data suggest a very-low-carbohydrate diet as a clinical tool to improve human T-cell immunity. Rethinking the value of nutrition and dietary interventions in modern medicine is required.

Background. Inflammation is an important driver of malignant glioma disease. Inflammatory mediators are not only produced by immune cells in the tumor microenvironment, but also by glioblastoma (GBM) cells themselves creating a mutually reinforcing loop. We here aimed at identifying an "anti-inflammatory switch" that allows to dampen inflammation in GBM. Methods. We used human GBM specimens, primary cultures, and cell lines. The response of GBM cells toward inflammatory stimuli was tested by incubation with supernatant of stimulated human immune cells. Expression levels were measured by whole transcriptome microarrays and qRT-PCR, and protein was quantified by LUMINEX and SDS-PAGE. MicroRNA binding to 3'UTRs was analyzed by luciferase assays. Proliferation rates were determined by flow cytometry, and invasion and angiogenesis were studied using migration and endothelial tube formation assays. Results. We demonstrated GBM cells to secrete high amounts of proinflammatory mediators in an inflammatory microenvironment. We found miR-93 as a potential "anti-inflammatory tumor suppressor" dramatically downregulated in GBM. Concordantly, cytokine secretion dropped after miR-93 re-expression. Transfection of miR-93 in GBM cells led to down-regulation of hubs of the inflammatory networks, namely, HIF-1 alpha and MAP3K2 as well as IL-6, G-CSF, IL-8, LIF, IL-1 ss, COX2, and CXCL5. We showed only COX2 and CXCL5 to be indirectly regulated by miR-93 while all other genes are true targets. Phenotypically, re-expression of miR-93 in GBM cells substantially suppressed proliferation, migration, and angiogenesis. Conclusions. Alleviating GBM-derived inflammation by re-expression of miR-93 may be a powerful tool to mitigate these tumors' aggressiveness and holds promise for new clinical approaches.

Background: The recombinant IL-1 receptor antagonist anakinra—currently approved for the treatment of autoinflammatory diseases—blocks IL-1β-mediated inflammatory signaling. As inflammation is a major driver of cancer, we hypothesized that anakinra might be able to mitigate glioblastoma (GBM) aggressiveness. Methods: Primary GBM or T98G cells were incubated alone or with peripheral blood mononuclear cells (PBMCs) and were subsequently treated with IL-1β and/or anakinra. T cells were obtained by magnetic bead isolation. Protein and mRNA expression were quantified by SDS-PAGE, qRT-PCR, and ELISA, respectively. Cell proliferation and apoptosis were analyzed via flow cytometry. Chemotaxis was studied via time-lapse microscopy. Results: Upon IL-1β stimulation, anakinra attenuated proinflammatory gene expression in both GBM cells and PBMCs, and mitigated tumor migration and proliferation. In a more lifelike model replacing IL-1β stimulation by GBM–PBMC co-culture, sole presence of PBMCs proved sufficient to induce a proinflammatory phenotype in GBM cells with enhanced proliferation and migration rates and attenuated apoptosis. Anakinra antagonized these pro-tumorigenic effects and, moreover, reduced inflammatory signaling in T cells without compromising anti-tumor effector molecules. Conclusion: By dampening the inflammatory crosstalk between GBM and immune cells, anakinra mitigated GBM aggressiveness. Hence, counteracting IL-1β-mediated inflammation might be a promising strategy to pursue.

Introduction Sepsis is defined as detrimental immune response to an infection. This overwhelming reaction often abolishes a normal reconstitution of the immune cell homeostasis that in turn increases the risk for further complications. Recent studies revealed a favourable impact of ketone bodies on resolution of inflammation. Thus, a ketogenic diet may provide an easy-to-apply and cost-effective treatment option potentially alleviating sepsis-evoked harm. This study is designed to assess the feasibility, efficiency and safety of a ketogenic diet in septic patients. Methods and analysis This monocentric study is a randomised, controlled and open-label trial, which is conducted on an intensive care unit of a German university hospital. As intervention enteral nutrition with reduced amount of carbohydrates (ketogenic) or standard enteral nutrition (control) is applied. The primary endpoint is the detection of ketone bodies in patients’ blood and urine samples. As secondary endpoints, the impact on important safety-relevant issues (eg, glucose metabolism, lactate serum concentration, incidence of metabolic acidosis, thyroid function and 30-day mortality) and the effect on the immune system are analysed. Ethics and dissemination The study has received the following approvals: Ethics Committee of the Medical Faculty of Ruhr-University Bochum (No. 18-6557-BR). Results will be made available to critical care survivors, their caregivers, the funders, the critical care societies and other researchers by publication in a peer-reviewed journal. Trial registration numbers German Clinical Trial Register (DRKS00017710); Universal Trial Number (U1111-1237-2493).

The recombinant IL-1 receptor antagonist anakinra prevents binding of IL-1 to its receptor and blocks IL-1β-mediated proinflammatory signaling. It is currently used in the treatment of autoinflammatory diseases. As inflammation is a major driver of malignancy, we hypothesized that anakinra – by ameliorating the inflammatory tumor environment – might be capable to mitigate glioblastoma (GBM) progression. T98G GBM cells were cultivated with PBMC, separated by cell culture inserts with or without anakinra and incubated 48h in 5% O2 mimicking the GBM microenvironment. T-cells were obtained by magnetic bead isolation. mRNA expression levels were measured by quantitative Real-Time-PCR (qRT-PCR). Cytokine production was assessed by ELISA. Cell proliferation was analyzed by flow cytometric quantification of Ki-67 protein expression. Anakinra has been provided by Swedish Orphan Biovitrum AB (Sweden). Results are presented as mean±SEM, p-Values were calculated using Student’s t-test. After administration of anakinra, mRNA expression levels of the inflammatory and proangiogenic genes IL-1β, COX-2, CCL2 and IL-8 were reduced in T98G (-64%±19.1%, -56.2%±21.3%, -87.1%±28.8%, -91.7%±27.2%, n=7, p< 0.05). Surprisingly, no changes of these targets were detectable in PBMC. However, in T-cells, anakinra induced mRNA expression of Th2 transcription factor GATA3 and the antiinflammatory cytokines IL-4 and IL-10 (+6,7%±4,6%, +36,1%±22,9%, +69,1%32,8%, n=10, p< 0.05). The expression of the protumorigenic genes IL-22, IL-17 as well as IFNγ was dramatically repressed (-63.2%±21.9%, -88.1%±21.7%, -85.6%±45.2%, n=10, p< 0.05), protein secretion of IL-22 and IFNγ was strongly blocked (-36.3%±9.1%, -13.9%±0.5%, n=6, p< 0.05). Importantly, treatment with anakinra markedly attenuated GBM cell proliferation (-20.1%±5%, n=4, p< 0.05). Anakinra indeed puts the brake on proinflammatory signaling pathways in GBM cells and simultaneously promotes a shift towards an anti-inflammatory T-cell phenotype. As a result, GBM proliferation is diminished. Hence, administration of anakinra might be an interesting and novel therapeutic approach to reduce Glioblastoma aggressiveness.

Complex immune dysregulation is a hallmark of sepsis. The occurring phases of immunosuppression and hyperinflammation require rapid detection and close monitoring. Reliable tools to monitor patient’s immune status are yet missing. Currently, microRNAs are being discussed as promising new biomarkers in sepsis. However, no suitable internal control for normalization of miRNA expression by qPCR has been validated so far, thus hampering their potential benefit. We here present the first evaluation of endogenous controls for miRNA analysis in human sepsis. Novel candidate reference miRNAs were identified via miRNA microArray. TaqMan qPCR assays were performed to evaluate these microRNAs in T-cells and whole blood cells of sepsis patients and healthy controls in two independent cohorts. In T-cells, U48 and miR-320 proved suitable as endogenous controls, while in whole blood cells, U44 and miR-942 provided best stability values for normalization of miRNA quantification. Commonly used snRNA U6 exhibited worst stability in all sample groups. The identified internal controls have been prospectively validated in independent cohorts. The critical importance of housekeeping gene selection is emphasized by exemplary quantification of imuno-miR-150 in sepsis patients. Use of appropriate internal controls could facilitate research on miRNA-based biomarker-use and might even improve treatment strategies in the future.

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.

Background: The second intron of Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4), an important hub in the pro-invasive MAPK pathway, harbors miR-744. There is accumulating evidence that intronic micro-RNAs (miRNAs) are capable of either supporting or restraining functional pathways of their host genes, thereby creating intricate regulative networks. We thus hypothesized that miR-744 regulates glioma migration by interacting with its host’s pathways. Methods: Patients’ tumor specimens were obtained stereotactically. MiR-744 was overexpressed in U87, T98G, and primary glioblastoma (GBM) cell lines. Cell mobility was studied using migration and Boyden chamber assays. Protein and mRNA expression was quantified by SDS-PAGE and qRT-PCR. Interactions of miR-744 and 3’UTRs were analyzed by luciferase reporter assays, and SMAD2/3, p38, and beta-Catenin activities by TOP/FOPflash reporter gene assays. Results: As compared to a normal brain, miR-744 levels were dramatically decreased in GBM samples and in primary GBM cell lines. Astrocytoma WHO grade II/III exhibited intermediate expression levels. Re-expression of miR-744 in U87, T98G, and primary GBM cell lines induced focal growth and impaired cell mobility. Luciferase activity of 3’UTR reporter constructs revealed the pro-invasive factors TGFB1 and DVL2 as direct targets of miR-744. Re-expression of miR-744 reduced levels of TGFB1, DVL2, and the host MAP2K4, and mitigated activity of TGFB1 and DVL2 downstream targets SMAD2/3 and beta-Catenin. TGFB1 knock-down repressed MAP2K4 expression. Conclusion: MiR-744 acts as an intrinsic brake on its host. It impedes MAP2K4 functional pathways through simultaneously targeting SMAD-, beta-Catenin, and MAPK signaling networks, thereby strongly mitigating pro-migratory effects of MAP2K4. MiR-744 is strongly repressed in glioma, and its re-expression might attenuate tumor invasiveness.

Differential expression of microRNAs (miRs) during inflammation and hypoxia is increasingly appreciated as central driver of glioblastoma (GBM) progression. As miR-93 inhibits expression Interleukin(IL)-8, we hypothesized that miR-93 might be a key regulator of inflammation within the GBM microenvironment. Stereotactically obtained GBM specimens were analysed by qRT-PCR. U87 cells were transfected with miR-93 by electroporation and subsequently incubated with IL1b in 5% O2 to mimic the GBM microenvironment. Whole transcriptome microarrays were processed and analyzed by bioinformatics. Invasion and angiogenesis were studied using U87 cell migration and endothelial tube formation assays. Expression levels were measured by qRT-PCR, multiplex assays, and SDS-PAGE (HIF1A, MAP3K2). Binding of miR-93 to 3`UTRs was analysed by luciferase reporter assays. Experiments were performed in triplicates at least 4 times. Results are presented as mean±SEM. P-Values were calculated using Student’s t-test. GBM specimens (n=33) and primary cell lines (n=5) revealed decreased levels of miR-93 compared to normal brain tissue (-55.6%±14% and -72.2%±22.3% p<0.05). Supernatants of miR-93-transfected U87 cells impaired U87 cell migration and attenuated angiogenesis of endothelial cells (migration:-58%±12.2%; angiogenesis:-62.4%±19.1%, n=5, p<0.05). Transfection of miR-93 resulted in strong repression of inflammatory genes as shown by transcriptome analyses and qRT-PCR (IL6:-89.6%±15.6%; IL8:-86.4%±16.5%; IL1b:-69.8%±13%; LIF:-59.7%±5.,9%, CSF3:-88.8%±17.9%; COX2:-76.3%±14.2%, CXCL5:-74.2%±13.8%, MAP3K2:-39.5%±20.9%; HIF1A:-15.4%±1.5%; n=4, p<0.05). Luciferase activity of five 3`UTR reporters were significantly quenched after miR-transfection (IL6:-29.2%±10.8%; LIF:-22%%±6.5%; CSF3:-31.8%±10.7%, MAP3K2:-45.3%±21.7%; HIF1A:-32.9%±8.4%, n=5, p<0.05), while COX2, CXCL5 exhibited no decrease. Combined knockdown of MAP3K2 and HIF1A significantly reduced COX2- and CXCL5 levels thus proving indirect regulation (-39%±4.3% and -25.9%±7.1%, respectively, n=8, p<0.05). These results suggest an anti-inflammatory and tumor suppressive role of miR-93. Through direct targeting of proinflammatory cytokines, MAP3K2, and HIF1A, miR-93 reduces tumor-initiated inflammation and inhibits shaping of a tumorigenic microenvironment. MiR-93 thus might be a promising target in new GBM treatment strategies.