Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma.

Adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell-intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor-reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network-based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

Exploring Valine Metabolism in Astrocytic and Liver Cells: Lesson from Clinical Observation in TBI Patients for Nutritional Intervention.

The utilization of alternative energy substrates to glucose could be beneficial in traumatic brain injury (TBI). Recent clinical data obtained in TBI patients reported valine, ß-hydroxyisobutyrate (ibHB) and 2-ketoisovaleric acid (2-KIV) as three of the main predictors of TBI outcome. In particular, higher levels of ibHB, 2-KIV, and valine in cerebral microdialysis (CMD) were associated with better clinical outcome. In this study, we investigate the correlations between circulating and CMD levels of these metabolites. We hypothesized that the liver can metabolize valine and provide a significant amount of intermediate metabolites, which can be further metabolized in the brain. We aimed to assess the metabolism of valine in human-induced pluripotent stem cell (iPSC)-derived astrocytes and HepG2 cells using 13C-labeled substrate to investigate potential avenues for increasing the levels of downstream metabolites of valine via valine supplementation. We observed that 94 ± 12% and 84 ± 16% of ibHB, and 94 ± 12% and 87 ± 15% of 2-KIV, in the medium of HepG2 cells and in iPSC-derived astrocytes, respectively, came directly from valine. Overall, these findings suggest that both ibHB and 2-KIV are produced from valine to a large extent in both cell types, which could be of interest in the design of optimal nutritional interventions aiming at stimulating valine metabolism.

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-ß (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.

The plant product quinic acid activates Ca2+ -dependent mitochondrial function and promotes insulin secretion from pancreatic beta cells.

BACKGROUND AND PURPOSE: Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. EXPERIMENTAL APPROACH: We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. KEY RESULTS: QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. CONCLUSIONS AND IMPLICATIONS: QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.

Differential Metabolism of Medium-Chain Fatty Acids in Differentiated Human-Induced Pluripotent Stem Cell-Derived Astrocytes.

Medium-chain triglyceride (MCT) ketogenic diets increase ketone bodies, which are believed to act as alternative energy substrates in the injured brain. Octanoic (C8:0) and decanoic (C10:0) acids, which produce ketone bodies through ß-oxidation, are used as part of MCT ketogenic diets. Although the ketogenic role of MCT is well-established, it remains unclear how the network metabolism underlying ß-oxidation of these medium-chain fatty acids (MCFA) differ. We aim to elucidate basal ß-oxidation of these commonly used MCFA at the cellular level. Human-induced pluripotent stem cell-derived (iPSC) astrocytes were incubated with [U-13C]-C8:0 or [U-13C]-C10:0, and the fractional enrichments (FE) of the derivatives were used for metabolic flux analysis. Data indicate higher extracellular concentrations and faster secretion rates of ß-hydroxybutyrate (ßHB) and acetoacetate (AcAc) with C8:0 than C10:0, and an important contribution from unlabeled substrates. Flux analysis indicates opposite direction of metabolic flux between the MCFA intermediates C6:0 and C8:0, with an important contribution of unlabeled sources to the elongation in the C10:0 condition, suggesting different ß-oxidation pathways. Finally, larger intracellular glutathione concentrations and secretions of 3-OH-C10:0 and C6:0 were measured in C10:0-treated astrocytes. These findings reveal MCFA-specific ketogenic properties. Our results provide insights into designing different MCT-based ketogenic diets to target specific health benefits.

Cellular stress is a prerequisite for glucose-induced mitochondrial matrix alkalinization in pancreatic ß-cells.

Changes in mitochondrial and cytosolic pH alter the chemical gradient across the inner mitochondrial membrane. The proton chemical gradient contributes to mitochondrial ATP synthesis as well as the uptake and release of metabolites and ions from the organelle. Here mitochondrial pH and ΔpH were studied for the first time in human pancreatic ß-cells. Adenoviruses were used for rat insulin promoter dependent expression of the pH sensor SypHer targeted to either the mitochondrial matrix or the cytosol. The matrix pH in resting human ß-cells is low (pH = 7.50 ±â€¯SD 0.17) compared to published values in other cell types. Consequently, the ΔpH of ß-cells mitochondria is small. Glucose stimulation consistently resulted in acidification of the matrix pH in INS-1E insulinoma cells and ß-cells in intact human islets or islet monolayer cultures. We registered acidification with similar kinetics but of slightly smaller amplitude in the cytosol of ß-cells, thus glucose stimulation further reduced the ΔpH. Infection of human islets with high levels of adenoviruses caused the mitochondrial pH to increase. The apoptosis inducer and broad-spectrum kinase inhibitor staurosporine had similar effects on pH homeostasis. Although staurosporine alone does not affect the mitochondrial pH, glucose slightly increases the matrix pH of staurosporine treated cells. These two cellular stressors alter the normal mitochondrial pH response to glucose in pancreatic ß-cells.

Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

In pancreatic ß-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in ß-cell energy metabolism and Ca2+ signaling.-De Marchi, U., Galindo, A. N., Thevenet, J., Hermant, A., Bermont, F., Lassueur, S., Domingo, J. S., Kussmann, M., Dayon, L., Wiederkehr, A. Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

A novel ATP-synthase-independent mechanism coupling mitochondrial activation to exocytosis in insulin-secreting cells.

Pancreatic ß-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in ß-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca2+ signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms.

Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 µM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 µM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

Calcium co-regulates oxidative metabolism and ATP synthase-dependent respiration in pancreatic beta cells.

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)(+) ratio.

Endogenous protease nexin-1 protects against cerebral ischemia.

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin's endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced ß-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1(-/-) mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.

Regulation of LRRK2 expression points to a functional role in human monocyte maturation.

Genetic variants of Leucine-Rich Repeat Kinase 2 (LRRK2) are associated with a significantly enhanced risk for Parkinson disease, the second most common human neurodegenerative disorder. Despite major efforts, our understanding of LRRK2 biological function and regulation remains rudimentary. In the present study we analyze LRRK2 mRNA and protein expression in sub-populations of human peripheral blood mononuclear cells (PBMCs). LRRK2 mRNA and protein was found in circulating CD19(+) B cells and in CD14(+) monocytes, whereas CD4(+) and CD8(+) T cells were devoid of LRRK2 mRNA. Within CD14(+) cells the CD14(+)CD16(+) sub-population of monocytes exhibited high levels of LRRK2 protein, in contrast to CD14(+)CD16(-) cells. However both populations expressed LRRK2 mRNA. As CD14(+)CD16(+) cells represent a more mature subset of monocytes, we monitored LRRK2 expression after in vitro treatment with various stress factors known to induce monocyte activation. We found that IFN-γ in particular robustly increased LRRK2 mRNA and protein levels in monocytes concomitant with a shift of CD14(+)CD16(-) cells towards CD14(+)CD16(+) cells. Interestingly, the recently described LRRK2 inhibitor IN-1 attenuated this shift towards CD14(+)CD16(+) after IFN-γ stimulation. Based on these findings we speculate that LRRK2 might have a role in monocyte maturation. Our results provide further evidence for the emerging role of LRRK2 in immune cells and regulation at the transcriptional and translational level. Our data might also reflect an involvement of peripheral and brain immune cells in the disease course of PD, in line with increasing awareness of the role of the immune system in PD.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Coagulation factor Xa activates thrombin in ischemic neural tissue.

Thrombin is involved in mediating neuronal death in cerebral ischemia. We investigated its so far unknown mode of activation in ischemic neural tissue. We used an in vitro approach to distinguish the role of circulating coagulation factors from endogenous cerebral mechanisms. We modeled ischemic stroke by subjecting rat organotypic hippocampal slice cultures to 30-min oxygen (5%) and glucose (1 mmol/L) deprivation (OGD). Perinuclear activated factor X (FXa) immunoreactivity was observed in CA1 neurons after OGD. Selective FXa inhibition by fondaparinux during and after OGD significantly reduced neuronal death in the CA1 after 48 h. Thrombin enzyme activity was increased in the medium 24 h after OGD and this increase was prevented by fondaparinux suggesting that FXa catalyzes the conversion of prothrombin to thrombin in neural tissue after ischemia in vitro. Treatment with SCH79797, a selective antagonist of the thrombin receptor protease-activated receptor-1 (PAR-1), significantly decreased neuronal cell death indicating that thrombin signals ischemic damage via PAR-1. The c-Jun N-terminal kinase (JNK) pathway plays an important role in excitotoxicity and cerebral ischemia and we observed activation of the JNK substrate, c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonist SCH79797, decreased the level of phospho-c-Jun Ser73. These results indicate that FXa activates thrombin in cerebral ischemia, which leads via PAR-1 to the activation of the JNK pathway resulting in neuronal death.

Neuroprotective role of lactate after cerebral ischemia.

It is well established that lactate can be used as an energy substrate by the brain by conversion to pyruvate and a subsequent oxidation in the mitochondria. Knowing the need for readily metabolizable substrates directly after ischemia and the protective effect of lactate after excitotoxicity, the aim of this study was to investigate whether lactate administration directly after ischemia could be neuroprotective. In vitro, the addition of 4 mmol/L L-lactate to the medium of rat organotypic hippocampal slices, directly after oxygen and glucose deprivation (OGD), protected against neuronal death, whereas a higher dose of 20 mmol/L was toxic. In vivo, after middle cerebral artery occlusion in the mouse, an intracerebroventricular injection of 2 microL of 100 mmol/L L-lactate, immediately after reperfusion, led to a significant decrease in lesion size, which was more pronounced in the striatum, and an improvement in neurologic outcome. A later injection 1 h after reperfusion did not reduce lesion size, but significantly improved neurologic outcome, which is an important point in the context of a potential clinical application. Therefore, a moderate increase in lactate after ischemia may be a therapeutic tool.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

D-JNKI1, a cell-penetrating c-Jun-N-terminal kinase inhibitor, protects against cell death in severe cerebral ischemia.

BACKGROUND AND PURPOSE: In 2 models of severe ischemic injury, we have evaluated the neuroprotective action of D-JNKI1, a cell-penetrating and protease-resistant peptide selectively inhibiting the c-Jun-N-terminal kinase (JNK). METHODS: Hippocampal slices from newborn rats were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation for 30 minutes. Cell death was evaluated with propidium iodide, and the evoked potential responses were recorded in the CA1 region after stimulation in CA3. Male ICR-CD1 mice were subjected to permanent endoluminal "suture" middle cerebral artery occlusion (MCAo). The lesion size was determined after 24 hours by triphenyl-tetrazolium chloride staining, and neurological scores and rotarod treadmill performance were used to evaluate the neurological outcome. RESULTS: In vitro, D-JNKI administration 6 hours after oxygen glucose deprivation reduced cell death at 24 hours from 21%+/-8% (n=10) to 5%+/-3% (n=7, P<0.01). This protective effect was still seen at 48 hours, paralleled by an improved amplitude of the evoked potential response. In vivo in the mouse, D-JNKI1 administration 3 hours after ischemia significantly reduced the infarct volume from 162+/-27 mm(3) (n=14) to 85+/-27 mm(3) (n=9, P<0.001). The functional outcome was also improved. CONCLUSIONS: JNK inhibition prevents cell death induced by oxygen and glucose deprivation in hippocampal slice cultures in vitro and by permanent suture MCAo in vivo. D-JNKI1 is a powerful neuroprotectant in models of both mild and severe cerebral ischemia, with an extended therapeutic window. Further investigations are needed to identify the relevant JNK target(s) mediating ischemic neuronal death.