Metabolomic and lipidomic signatures associated with activation of human cDC1 (BDCA3+ /CD141+ ) dendritic cells.

Dendritic cells (DCs) bridge the connection between innate and adaptive immunity. DCs present antigens to T cells and stimulate potent cytotoxic T-cell responses. Metabolic reprogramming is critical for DC development and activation; however, metabolic adaptations and regulation in DC subsets remains largely uncharacterized. Here, we mapped metabolomic and lipidomic signatures associated with the activation phenotype of human conventional DC type 1, a DC subset specialized in cross-presentation and therefore of major importance for the stimulation of CD8+ T cells. Our metabolomics and lipidomic analyses showed that Toll-like receptor (TLR) stimulation altered glycerolipids and amino acids in cDC1. Poly I:C or pRNA stimulation reduced triglycerides and cholesterol esters, as well as various amino acids. Moreover, TLR stimulation reduced expression of glycolysis-regulating genes and did not induce glycolysis. Conversely, cDC1 exhibited increased mitochondrial content and oxidative phosphorylation (OXPHOS) upon TLR3 or TLR7/8 stimulation. Our findings highlight the metabolic adaptations required for cDC1 maturation.

Mitochondria-targeted phenolic antioxidants induce ROS-protective pathways in primary human skin fibroblasts.

Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondria-targeted variants of gallic acid (AntiOxBEN2) and caffeic acid (AntiOxCIN4). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 ~23 µM). At a relatively high but non-toxic concentration (12.5 µM), AntiOxBEN2 and AntiOxCIN4 increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN2 and AntiOxCIN4 did not alter mitochondrial membrane potential (Δψ), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P)H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN2 and AntiOxCIN4 both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN2 (but not AntiOxCIN4) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN2 and AntiOxCIN4 increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN2 and AntiOxCIN4 can act as prooxidants thereby activating endogenous ROS-protective pathways.

Dendritic Cells Require PINK1-Mediated Phosphorylation of BCKDE1α to Promote Fatty Acid Oxidation for Immune Function.

Dendritic cell (DCs) activation by Toll-like receptor (TLR) agonist induces robust metabolic rewiring toward glycolysis. Recent findings in the field identified mechanistic details governing these metabolic adaptations. However, it is unknown whether a switch to glycolysis from oxidative phosphorylation (OXPHOS) is a general characteristic of DCs upon pathogen encounter. Here we show that engagement of different TLR triggers differential metabolic adaptations in DCs. We demonstrate that LPS-mediated TLR4 stimulation induces glycolysis in DCs. Conversely, activation of TLR7/8 with protamine-RNA complex, pRNA, leads to an increase in OXPHOS. Mechanistically, we found that pRNA stimulation phosphorylates BCKDE1α in a PINK1-dependent manner. pRNA stimulation increased branched-chain amino acid levels and increased fatty acid oxidation. Increased FAO and OXPHOS are required for DC activation. PINK1 deficient DCs switch to glycolysis to maintain ATP levels and viability. Moreover, pharmacological induction of PINK1 kinase activity primed immunosuppressive DC for immunostimulatory function. Our findings provide novel insight into differential metabolic adaptations and reveal the important role of branched-chain amino acid in regulating immune response in DC.

Human Dendritic Cell Subsets Undergo Distinct Metabolic Reprogramming for Immune Response.

Toll-like receptor (TLR) agonists induce metabolic reprogramming, which is required for immune activation. We have investigated mechanisms that regulate metabolic adaptation upon TLR-stimulation in human blood DC subsets, CD1c+ myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). We show that TLR-stimulation changes expression of genes regulating oxidative phosphorylation (OXPHOS) and glutamine metabolism in pDC. TLR-stimulation increases mitochondrial content and intracellular glutamine in an autophagy-dependent manner in pDC. TLR-induced glutaminolysis fuels OXPHOS in pDCs. Notably, inhibition of glutaminolysis and OXPHOS prevents pDC activation. Conversely, TLR-stimulation reduces mitochondrial content, OXPHOS activity and induces glycolysis in CD1c+ mDC. Inhibition of mitochondrial fragmentation or promotion of mitochondrial fusion impairs TLR-stimulation induced glycolysis and activation of CD1c+ mDCs. TLR-stimulation triggers BNIP3-dependent mitophagy, which regulates transcriptional activity of AMPKα1. BNIP3-dependent mitophagy is required for induction of glycolysis and activation of CD1c+ mDCs. Our findings reveal that TLR stimulation differentially regulates mitochondrial dynamics in distinct human DC subsets, which contributes to their activation.

The Myc/Max/Mxd Network Is a Target of Mutated Flt3 Signaling in Hematopoietic Stem Cells in Flt3-ITD-Induced Myeloproliferative Disease.

Acute myeloid leukemia (AML) has poor prognosis due to various mutations, e.g., in the FLT3 gene. Therefore, it is important to identify pathways regulated by the activated Flt3 receptor for the discovery of new therapeutic targets. The Myc network of oncogenes and tumor suppressor genes is involved in mechanisms regulating proliferation and survival of cells, including that of the hematopoietic system. In this study, we evaluated the expression of the Myc oncogenes and Mxd antagonists in hematopoietic stem cell and myeloid progenitor populations in the Flt3-ITD-knockin myeloproliferative mouse model. Our data shows that the expression of Myc network genes is changed in Flt3-ITD mice compared with the wild type. Mycn is increased in multipotent progenitors and in the pre-GM compartment of myeloid progenitors in the ITD mice while the expression of several genes in the tumor suppressor Mxd family, including Mxd1, Mxd2, and Mxd4, is concomitantly downregulated, as well as the expression of the Mxd-related gene Mnt and the transcriptional activator Miz-1. LSKCD150+CD48- hematopoietic long-term stem cells are decreased in the Flt3-ITD cells while multipotent progenitors are increased. Of note, PKC412-mediated inhibition of Flt3-ITD signaling results in downregulation of cMyc and upregulation of the Myc antagonists Mxd1, Mxd2, and Mxd4. Our data provides new mechanistic insights into downstream alterations upon aberrant Flt3 signaling and rationale for combination therapies for tyrosine kinase inhibitors with Myc antagonists in treating AML.

Extracellular acidification induces ROS- and mPTP-mediated death in HEK293 cells.

The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Δψ), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant α-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism.

Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells.

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 ('BAY') triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.

Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells.

Inhibitor studies with isolated mitochondria demonstrated that complex I (CI) and III (CIII) of the electron transport chain (ETC) can act as relevant sources of mitochondrial reactive oxygen species (ROS). Here we studied ROS generation and oxidative stress induction during chronic (24h) inhibition of CI and CIII using rotenone (ROT) and antimycin A (AA), respectively, in intact HEK293 cells. Both inhibitors stimulated oxidation of the ROS sensor hydroethidine (HEt) and increased mitochondrial NAD(P)H levels without major effects on cell viability. Integrated analysis of cells stably expressing cytosolic- or mitochondria-targeted variants of the reporter molecules HyPer (H2O2-sensitive and pH-sensitive) and SypHer (H2O2-insensitive and pH-sensitive), revealed that CI- and CIII inhibition increased cytosolic but not mitochondrial H2O2 levels. Total and mitochondria-specific lipid peroxidation was not increased in the inhibited cells as reported by the C11-BODIPY(581/591) and MitoPerOx biosensors. Also expression of the superoxide-detoxifying enzymes CuZnSOD (cytosolic) and MnSOD (mitochondrial) was not affected. Oxyblot analysis revealed that protein carbonylation was not stimulated by CI and CIII inhibition. Our findings suggest that chronic inhibition of CI and CIII: (i) increases the levels of HEt-oxidizing ROS and (ii) specifically elevates cytosolic but not mitochondrial H2O2 levels, (iii) does not induce oxidative stress or substantial cell death. We conclude that the increased ROS levels are below the stress-inducing level and might play a role in redox signaling.

Targeting mitochondrial complex I using BAY 87-2243 reduces melanoma tumor growth.

BACKGROUND: Numerous studies have demonstrated that functional mitochondria are required for tumorigenesis, suggesting that mitochondrial oxidative phosphorylation (OXPHOS) might be a potential target for cancer therapy. In this study, we investigated the effects of BAY 87-2243, a small molecule that inhibits the first OXPHOS enzyme (complex I), in melanoma in vitro and in vivo. RESULTS: BAY 87-2243 decreased mitochondrial oxygen consumption and induced partial depolarization of the mitochondrial membrane potential. This was associated with increased reactive oxygen species (ROS) levels, lowering of total cellular ATP levels, activation of AMP-activated protein kinase (AMPK), and reduced cell viability. The latter was rescued by the antioxidant vitamin E and high extracellular glucose levels (25 mM), indicating the involvement of ROS-induced cell death and a dependence on glycolysis for cell survival upon BAY 87-2243 treatment. BAY 87-2243 significantly reduced tumor growth in various BRAF mutant melanoma mouse xenografts and patient-derived melanoma mouse models. Furthermore, we provide evidence that inhibition of mutated BRAF using the specific small molecule inhibitor vemurafenib increased the OXPHOS dependency of BRAF mutant melanoma cells. As a consequence, the combination of both inhibitors augmented the anti-tumor effect of BAY 87-2243 in a BRAF mutant melanoma mouse xenograft model. CONCLUSIONS: Taken together, our results suggest that complex I inhibition has potential clinical applications as a single agent in melanoma and also might be efficacious in combination with BRAF inhibitors in the treatment of patients with BRAF mutant melanoma.

RIP1 protein-dependent assembly of a cytosolic cell death complex is required for inhibitor of apoptosis (IAP) inhibitor-mediated sensitization to lexatumumab-induced apoptosis.

Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death.