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Sodium chloride promotes vascular fibrosis, arterial hypertension, pro-inflammatory immune cell polarization and endothelial dysfunction, all of which might influence outcomes following stroke. But despite enormous translational relevance, the functional importance of sodium chloride in the pathophysiology of acute ischemic stroke is still unclear. In the current study, we show that high-salt diet leads to significantly worse functional outcomes, increased infarct volumes, and a loss of astrocytes and cortical neurons in acute ischemic stroke. While analyzing the underlying pathologic processes, we identified the migrasome as a novel, sodium chloride-driven pathomechanism in acute ischemic stroke. The migrasome was previously described in vitro as a migrating organelle, which incorporates and dispatches cytosol of surrounding cells and plays a role in intercellular signaling, whereas a pathophysiological meaning has not been elaborated. We here confirm previously reported characteristics of the migrasome in vivo. Immunohistochemistry, electron microscopy and proteomic analyses further demonstrate that the migrasome incorporates and dispatches cytosol of surrounding neurons following stroke. The clinical relevance of these findings is emphasized by neuropathological examinations, which detected migrasome formation in infarcted brain parenchyma of human stroke patients. In summary, we demonstrate that high-salt diet aggravates stroke outcomes, and we characterize the migrasome as a novel mechanism in acute stroke pathophysiology.

The concept of trained innate immunity describes a long-term proinflammatory memory in innate immune cells. Trained innate immunity is regulated through reprogramming of cellular metabolic pathways including cholesterol and fatty acid synthesis. Here, we have analyzed the role of Liver X Receptor (LXR), a key regulator of cholesterol and fatty acid homeostasis, in trained innate immunity. Human monocytes were isolated and incubated with different stimuli for 24 h, including LXR agonists, antagonists and Bacillus Calmette-Guerin (BCG) vaccine. After 5 days resting time, cells were restimulated with the TLR2-agonist Pam3cys. LXR activation did not only increase BCG trained immunity, but also induced a long-term inflammatory activation by itself. This inflammatory activation by LXR agonists was accompanied by characteristic features of trained innate immunity, such as activating histone marks on inflammatory gene promoters and metabolic reprogramming with increased lactate production and decreased oxygen consumption rate. Mechanistically, LXR priming increased cellular acetyl-CoA levels and was dependent on the activation of the mevalonate pathway and IL-1β signaling. In contrast to mevalonate pathway inhibition, blocking fatty acid synthesis further increased proinflammatory priming by LXR. We demonstrate that LXR activation induces a proinflammatory trained immunity phenotype in human monocytes through epigenetic and metabolic reprogramming. Our data reveal important novel aspects of LXR signaling in innate immunity.

Recent studies highlighted the role of transcription factors in metabolic regulation during hematopoiesis and leukemia development. GFI1B is a transcriptional repressor that plays a critical role in hematopoiesis, and its expression is negatively related to the prognosis of acute myeloid leukemia (AML) patients. We earlier reported a change in the metabolic state of hematopoietic stem cells upon Gfi1b deletion. Here we explored the role of Gfi1b in metabolism reprogramming during hematopoiesis and leukemogenesis. We demonstrated that Gfi1b deletion remarkably activated mitochondrial respiration and altered energy metabolism dependence toward oxidative phosphorylation (OXPHOS). Mitochondrial substrate dependency was shifted from glucose to fatty acids upon Gfi1b deletion via upregulating fatty acid oxidation (FAO). On a molecular level, Gfi1b epigenetically regulated multiple FAO-related genes. Moreover, we observed that metabolic phenotypes evolved as cells progressed from preleukemia to leukemia, and the correlation between Gfi1b expression level and metabolic phenotype was affected by genetic variations in AML cells. FAO or OXPHOS inhibition significantly impeded leukemia progression of Gfi1b-KO MLL/AF9 cells. Finally, we showed that Gfi1b-deficient AML cells were more sensitive to metformin as well as drugs implicated in OXPHOS and FAO inhibition, opening new potential therapeutic strategies.

Background The nuclear receptor farnesoid-X-receptor (FXR; NR1H4) is expressed not only in the liver, gut, kidney and adipose tissue but also in the immune cells. FXR has been shown to confer protection in several animal models of inflammation, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). FXR agonists are currently tested in clinical trials for treatment of human metabolic diseases. The beneficial effect of FXR agonists in EAE suggests that FXR might represent a potential target in inflammatory-demyelinating CNS diseases, such as MS. In MS, oligodendrocytes not only undergo cell death but also contribute to remyelination. This repair mechanism is impaired due to a differentiation block of oligodendroglial progenitor cells. Activation of other nuclear receptors that heterodimerize with FXR promote oligodendroglial differentiation. Therefore, we wanted to address the functional relevance of FXR for glial cells, especially for oligodendroglial differentiation. Methods We isolated primary murine oligodendrocytes from FXR-deficient (FXR Ko) and wild-type (WT) mice and determined the effect of FXR deficiency and activation on oligodendroglial differentiation by analysing markers of oligodendroglial progenitor cells (OPCs) and mature oligodendrocytes (OLs) using qRT-PCR and immunocytochemistry. Additionally, we determined whether FXR activation modulates the pro-inflammatory profile of astrocytes or microglia and whether this may subsequently modulate oligodendroglial differentiation. These in vitro studies were complemented by histological analyses of oligodendrocytes in FXR Ko mice. Results FXR is expressed by OPCs and mature oligodendrocytes. However, lack of FXR did not affect oligodendroglial differentiation in vitro or in vivo. Furthermore, activation of FXR using the synthetic agonist GW4064 did not affect oligodendroglial differentiation, remyelination in an ex vivo model or the expression of pro-inflammatory molecules in astrocytes or microglia. Concordantly, no effects of supernatants from macrophages cultured in the presence of GW4064 were observed regarding a possible indirect impact on oligodendroglial differentiation. Conclusions Our data suggest that FXR is dispensable for oligodendroglial differentiation and that FXR agonists, such as GW4064, represent a potential therapeutic approach for MS which specifically targets peripheral immune cells including macrophages but not brain-resident cells, such as oligodendrocytes, astrocytes or microglia.

T cells critically depend on reprogramming of metabolic signatures to meet the bioenergetic demands during activation and clonal expansion. Here we identify the transcription factor Nur77 as a cell-intrinsic modulator of T cell activation. Nur77-deficient T cells are highly proliferative, and lack of Nur77 is associated with enhanced T cell activation and increased susceptibility for T cell-mediated inflammatory diseases, such as CNS autoimmunity, allergic contact dermatitis and collagen-induced arthritis. Importantly, Nur77 serves as key regulator of energy metabolism in T cells, restricting mitochondrial respiration and glycolysis and controlling switching between different energy pathways. Transcriptional network analysis revealed that Nur77 modulates the expression of metabolic genes, most likely in close interaction with other transcription factors, especially estrogen-related receptor α. In summary, we identify Nur77 as a transcriptional regulator of T cell metabolism, which elevates the threshold for T cell activation and confers protection in different T cell-mediated inflammatory diseases.

Acute myeloid leukemia (AML) is a group of hematological cancers with metabolic heterogeneity. Oxidative phosphorylation (OXPHOS) has been reported to play an important role in the function of leukemic stem cells and chemotherapy-resistant cells and are associated with inferior prognosis in AML patients. However, the relationship between metabolic phenotype and genetic mutations are yet to be explored. In the present study, we demonstrate that AML cell lines have high metabolic heterogeneity, and AML cells with MLL/AF9 have upregulated mitochondrial activity and mainly depend on OXPHOS for energy production. Furthermore, we show that metformin repressed the proliferation of MLL/AF9 AML cells by inhibiting mitochondrial respiration. Together, this study demonstrates that AML cells with an MLL/AF9 genotype have a high dependency on OXPHOS and could be therapeutically targeted by metformin.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell’s metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy- d -glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N -linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme’s function from transcription to replication.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease causing axonal degeneration and demyelination. Exercise in mice with active monophasic experimental autoimmune encephalomyelitis (EAE) attenuates disease severity associated with diverse impacts on T cell-mediated immunity. However, studies have so far focused on preventive approaches. In this study, we investigated the impact of endurance exercise on established EAE disease in a model of secondary progressive MS. When the exercise program on motorized running wheels was started at disease manifestation, the disease course was significantly ameliorated. This was associated with a significant decrease in B cell, dendritic cell, and neutrophil cell counts in the central nervous system (CNS). Furthermore, we observed an increased expression of major histocompatibility complex class II (MHC-II) as well as alterations in costimulatory molecule expression in CNS B cells and dendritic cells. In contrast, T cell responses were not altered in the CNS or periphery. Thus, exercise training is capable of attenuating the disease course even in established secondary progressive EAE, potentially via modulation of the innate immune compartment. Further studies are warranted to corroborate our findings and assess the potential of this lifestyle intervention as a complementary therapeutic strategy in secondary progressive MS patients.

Pericytes at the blood–brain barrier (BBB) are located between the tight endothelial cell layer of the blood vessels and astrocytic endfeet. They contribute to central nervous system (CNS) homeostasis by regulating BBB development and maintenance. Loss of pericytes results in increased numbers of infiltrating immune cells in the CNS in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis (MS). However, little is known about their competence to modulate immune cell activation or function in CNS autoimmunity. To evaluate the capacity of pericytes to directly interact with T cells in an antigen-specific fashion and potentially (re)shape their function, we depleted major histocompatibility complex (MHC) class II from pericytes in a cell type-specific fashion and performed T cell-pericyte cocultures and EAE experiments. We found that pericytes present antigen in vitro to induce T cell activation and proliferation. In an adoptive transfer EAE experiment, pericyte-specific MHC II KO resulted in locally enhanced T cell infiltration in the CNS; even though, overall disease course of mice was not affected. Thus, pericytes may serve as non-professional antigen-presenting cells affecting states of T cell activation, thereby locally shaping lesion formation in CNS inflammation but without modulating disease severity.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca 2+ ) is the most important second messenger, for which the potassium channel K 2P 18.1 is a relevant regulator. Here, we identify K 2P 18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K 2P 18.1 upregulation in tTreg progenitors. K 2P 18.1 provided the driving force for sustained Ca 2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K 2P 18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K 2P 18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K 2P 18.1 variant that is associated with poor clinical outcomes indicate that K 2P 18.1 also plays a role in human Treg development. Pharmacological modulation of K 2P 18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K 2P 18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K 2P 18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.