bims-mepmim 2024-05-26 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2024‒05‒26
twenty papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Mitochondrial respiratory function is preserved under cysteine starvation via glutathione catabolism in NSCLC.
Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.
Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.
Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice.
PPA1 promotes adipogenesis by regulating the stability of C/EBPs.
Metabolic coordination between skin epithelium and type 17 immunity sustains chronic skin inflammation.
Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.
Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.
Protective role for kidney TREM2high macrophages in obesity- and diabetes-induced kidney injury.
DHCR24 inhibitor SH42 increases desmosterol without preventing atherosclerosis development in mice.
Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice.
A metabolic dependency of EBV can be targeted to hinder B cell transformation.
Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys.
Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.
Preserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG.
Production of deoxycholic acid by low-abundant microbial species is associated with impaired glucose metabolism.
A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.
Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.
High nitrate levels in skeletal muscle contribute to nitric oxide generation via a nitrate/nitrite reductive pathway in mice that lack the nNOS enzyme.
Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis.

Nat Commun. 2024 May 18. 15(1): 4244

Mitochondrial respiratory function is preserved under cysteine starvation via glutathione catabolism in NSCLC.

Nathan P Ward, Sang Jun Yoon, Tyce Flynn, Amanda M Sherwood, Maddison A Olley, Juliana Madej, Gina M DeNicola.

Cysteine metabolism occurs across cellular compartments to support diverse biological functions and prevent the induction of ferroptosis. Though the disruption of cytosolic cysteine metabolism is implicated in this form of cell death, it is unknown whether the substantial cysteine metabolism resident within the mitochondria is similarly pertinent to ferroptosis. Here, we show that despite the rapid depletion of intracellular cysteine upon loss of extracellular cystine, cysteine-dependent synthesis of Fe-S clusters persists in the mitochondria of lung cancer cells. This promotes a retention of respiratory function and a maintenance of the mitochondrial redox state. Under these limiting conditions, we find that glutathione catabolism by CHAC1 supports the mitochondrial cysteine pool to sustain the function of the Fe-S proteins critical to oxidative metabolism. We find that disrupting Fe-S cluster synthesis under cysteine restriction protects against the induction of ferroptosis, suggesting that the preservation of mitochondrial function is antagonistic to survival under starved conditions. Overall, our findings implicate mitochondrial cysteine metabolism in the induction of ferroptosis and reveal a mechanism of mitochondrial resilience in response to nutrient stress.

DOI: https://doi.org/10.1038/s41467-024-48695-2
Nat Commun. 2024 May 24. 15(1): 4444

Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies.

Meng-Han Wu, Felipe Valenca-Pereira, Francesca Cendali, Emily L Giddings, Catherine Pham-Danis, Michael C Yarnell, Amanda J Novak, Tonya M Brunetti, Scott B Thompson, Jorge Henao-Mejia, Richard A Flavell, Angelo D'Alessandro, M Eric Kohler, Mercedes Rincon.

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

DOI: https://doi.org/10.1038/s41467-024-48653-y
Redox Biol. 2024 May 13. pii: S2213-2317(24)00169-1. [Epub ahead of print]73 103191

Distinct metabolic responses to heme in inflammatory human and mouse macrophages - Role of nitric oxide.

Pooja Pradhan, Vijith Vijayan, Bin Liu, Beatriz Martinez-Delgado, Nerea Matamala, Christoph Nikolin, Robert Greite, David S DeLuca, Sabina Janciauskiene, Roberto Motterlini, Roberta Foresti, Stephan Immenschuh.

  Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Keywords:  Heme; Inflammation; Lipopolysaccharide; Macrophages; Mitochondrial metabolism; Nitric oxide

DOI:  https://doi.org/10.1016/j.redox.2024.103191
Nat Commun. 2024 May 23. 15(1): 4393

Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice.

Yuan Liang, Chao Luo, Lijun Sun, Tiange Feng, Wenzhen Yin, Yunhua Zhang, Michael W Mulholland, Weizhen Zhang, Yue Yin.

Whether intestinal Leucine-rich repeat containing G-protein-coupled receptor 4 (LGR4) impacts nutrition absorption and energy homeostasis remains unknown. Here, we report that deficiency of Lgr4 (Lgr4iKO) in intestinal epithelium decreased the proportion of enterocytes selective for long-chain fatty acid absorption, leading to reduction in lipid absorption and subsequent improvement in lipid and glucose metabolism. Single-cell RNA sequencing demonstrates the heterogeneity of absorptive enterocytes, with a decrease in enterocytes selective for long-chain fatty acid-absorption and an increase in enterocytes selective for carbohydrate absorption in Lgr4iKO mice. Activation of Notch signaling and concurrent inhibition of Wnt signaling are observed in the transgenes. Associated with these alterations is the substantial reduction in lipid absorption. Decrement in lipid absorption renders Lgr4iKO mice resistant to high fat diet-induced obesity relevant to wild type littermates. Our study thus suggests that targeting intestinal LGR4 is a potential strategy for the intervention of obesity and liver steatosis.

DOI: https://doi.org/10.1038/s41467-024-48622-5
Cell Death Differ. 2024 May 18.

PPA1 promotes adipogenesis by regulating the stability of C/EBPs.

Yangyang Wu, Yue Sun, Yuqing Song, Jiateng Wang, Ye Han, Nan Yang, Haiyan Lin, Ye Yin, Xiao Han.

Adipogenesis significantly contributes to healthy adipose tissue expansion in obesity. Increasing adipocyte number or function to alleviate adipose tissue overload could serve as a therapeutic strategy for both lipodystrophy and obesity-related metabolic syndrome. Inorganic pyrophosphatase (PPA1) is an enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) and is involved in many biochemical reactions, but its function in adipose tissue has not been studied previously. In this study, we demonstrated that adipose-specific PPA1 knockout (PPA1AKO) mice showed lipodystrophy and spontaneously developed hepatic steatosis and severe insulin resistance under normal chow diet feeding. PPA1 deficiency suppressed the differentiation of primary adipocyte precursors and 3T3-L1 cells. Notably, PPA1 overexpression can restore inhibited adipogenesis in preadipocytes isolated from db/db mice and type 2 diabetes patients. Mechanistic studies have revealed that PPA1 acts as a positive regulator of early adipocyte differentiation by promoting CCAAT/enhancer-binding proteinβ and δ (C/EBPβ and δ) protein stability. Moreover, the function of PPA1 in adipogenesis is independent of its PPi catalytic activity. Collectively, our in vivo and in vitro findings demonstrated that PPA1 is a novel critical upstream regulator of adipogenesis, controlling adipose tissue development and whole-body metabolic homeostasis.

DOI: https://doi.org/10.1038/s41418-024-01309-2
Immunity. 2024 May 14. pii: S1074-7613(24)00227-9. [Epub ahead of print]

Metabolic coordination between skin epithelium and type 17 immunity sustains chronic skin inflammation.

Ipsita Subudhi, Piotr Konieczny, Aleksandr Prystupa, Rochelle L Castillo, Erica Sze-Tu, Yue Xing, Daniel Rosenblum, Ilana Reznikov, Ikjot Sidhu, Cynthia Loomis, Catherine P Lu, Niroshana Anandasabapathy, Mayte Suárez-Fariñas, Johann E Gudjonsson, Aristotelis Tsirigos, Jose U Scher, Shruti Naik.

  Inflammatory epithelial diseases are spurred by the concomitant dysregulation of immune and epithelial cells. How these two dysregulated cellular compartments simultaneously sustain their heightened metabolic demands is unclear. Single-cell and spatial transcriptomics (ST), along with immunofluorescence, revealed that hypoxia-inducible factor 1α (HIF1α), downstream of IL-17 signaling, drove psoriatic epithelial remodeling. Blocking HIF1α in human psoriatic lesions ex vivo impaired glycolysis and phenocopied anti-IL-17 therapy. In a murine model of skin inflammation, epidermal-specific loss of HIF1α or its target gene, glucose transporter 1, ameliorated epidermal, immune, vascular, and neuronal pathology. Mechanistically, glycolysis autonomously fueled epithelial pathology and enhanced lactate production, which augmented the γδ T17 cell response. RORγt-driven genetic deletion or pharmacological inhibition of either lactate-producing enzymes or lactate transporters attenuated epithelial pathology and IL-17A expression in vivo. Our findings identify a metabolic hierarchy between epithelial and immune compartments and the consequent coordination of metabolic processes that sustain inflammatory disease.

Keywords:  HIF1α; epithelium; glycolysis; inflammation; lactate; metabolism; skin; type 17 cells

DOI:  https://doi.org/10.1016/j.immuni.2024.04.022
Elife. 2024 May 22. pii: RP89377. [Epub ahead of print]12

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Tobias Weinberger, Messerer Denise, Markus Joppich, Maximilian Fischer, Clarisabel Garcia Rodriguez, Konda Kumaraswami, Vanessa Wimmler, Sonja Ablinger, Saskia Räuber, Jiahui Fang, Lulu Liu, Wing Han Liu, Julia Winterhalter, Johannes Lichti, Lukas Thomas, Dena Esfandyari, Guelce Percin, Sandra Matin, Andrés Hidalgo, Claudia Waskow, Stefan Engelhardt, Andrei Todica, Ralf Zimmer, Clare Pridans, Elisa Gomez Perdiguero, Christian Schulz.

  Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

Keywords:  developmental biology; immunology; inflammation; macrophage; mouse; myocardial infarction

DOI:  https://doi.org/10.7554/eLife.89377
Elife. 2024 May 24. pii: RP95652. [Epub ahead of print]13

Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.

Keene L Abbott, Ahmed Ali, Bradley I Reinfeld, Amy Deik, Sonu Subudhi, Madelyn D Landis, Rachel A Hongo, Kirsten L Young, Tenzin Kunchok, Christopher S Nabel, Kayla D Crowder, Johnathan R Kent, Maria Lucia L Madariaga, Rakesh K Jain, Kathryn E Beckermann, Caroline A Lewis, Clary B Clish, Alexander Muir, W Kimryn Rathmell, Jeffrey Rathmell, Matthew G Vander Heiden.

  The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer-driven tissue factors in shaping nutrient availability in these tumors.

Keywords:  cancer; cancer biology; human; metabolism; tumor microenvironment

DOI:  https://doi.org/10.7554/eLife.95652
Cell Rep. 2024 May 21. pii: S2211-1247(24)00581-3. [Epub ahead of print]43(6): 114253

Protective role for kidney TREM2high macrophages in obesity- and diabetes-induced kidney injury.

Ayshwarya Subramanian, Katherine A Vernon, Yiming Zhou, Jamie L Marshall, Maria Alimova, Carlos Arevalo, Fan Zhang, Michal Slyper, Julia Waldman, Monica S Montesinos, Danielle Dionne, Lan T Nguyen, Michael S Cuoco, Dan Dubinsky, Jason Purnell, Keith Keller, Samuel H Sturner, Elizabeth Grinkevich, Ayan Ghoshal, Amanda Kotek, Giorgio Trivioli, Nathan Richoz, Mary B Humphrey, Isabella G Darby, Sarah J Miller, Yingping Xu, Astrid Weins, Alexandra Chloe-Villani, Steven L Chang, Matthias Kretzler, Orit Rosenblatt-Rosen, Jillian L Shaw, Kurt A Zimmerman, Menna R Clatworthy, Aviv Regev, Anna Greka.

  Diabetic kidney disease (DKD), the most common cause of kidney failure, is a frequent complication of diabetes and obesity, and yet to date, treatments to halt its progression are lacking. We analyze kidney single-cell transcriptomic profiles from DKD patients and two DKD mouse models at multiple time points along disease progression-high-fat diet (HFD)-fed mice aged to 90-100 weeks and BTBR ob/ob mice (a genetic model)-and report an expanding population of macrophages with high expression of triggering receptor expressed on myeloid cells 2 (TREM2) in HFD-fed mice. TREM2high macrophages are enriched in obese and diabetic patients, in contrast to hypertensive patients or healthy controls in an independent validation cohort. Trem2 knockout mice on an HFD have worsening kidney filter damage and increased tubular epithelial cell injury, all signs of worsening DKD. Together, our studies suggest that strategies to enhance kidney TREM2high macrophages may provide therapeutic benefits for DKD.

Keywords:  CP: Immunology; TREM2; diabetic kidney disease; immune; inflammation; macrophage; myeloid; single-cell transcriptomics

DOI:  https://doi.org/10.1016/j.celrep.2024.114253
iScience. 2024 Jun 21. 27(6): 109830

DHCR24 inhibitor SH42 increases desmosterol without preventing atherosclerosis development in mice.

Xiaoke Ge, Bram Slütter, Joost M Lambooij, Enchen Zhou, Zhixiong Ying, Ceren Agirman, Marieke Heijink, Antoine Rimbert, Bruno Guigas, Johan Kuiper, Christoph Müller, Franz Bracher, Martin Giera, Sander Kooijman, Patrick C N Rensen, Yanan Wang, Milena Schönke.

  The liver X receptor (LXR) is considered a therapeutic target for atherosclerosis treatment, but synthetic LXR agonists generally also cause hepatic steatosis and hypertriglyceridemia. Desmosterol, a final intermediate in cholesterol biosynthesis, has been identified as a selective LXR ligand that suppresses inflammation without inducing lipogenesis. Δ24-Dehydrocholesterol reductase (DHCR24) converts desmosterol into cholesterol, and we previously showed that the DHCR24 inhibitor SH42 increases desmosterol to activate LXR and attenuate experimental peritonitis and metabolic dysfunction-associated steatotic liver disease. Here, we aimed to evaluate the effect of SH42 on atherosclerosis development in APOE∗3-Leiden.CETP mice and low-density lipoproteins (LDL) receptor knockout mice, models for lipid- and inflammation-driven atherosclerosis, respectively. In both models, SH42 increased desmosterol without affecting plasma lipids. While reducing liver lipids in APOE∗3-Leiden.CETP mice, and regulating populations of circulating monocytes in LDL receptor knockout mice, SH42 did not attenuate atherosclerosis in either model.

Keywords:  Cardiovascular medicine; Cellular physiology; Molecular genetics;

DOI:  https://doi.org/10.1016/j.isci.2024.109830
Redox Biol. 2024 May 17. pii: S2213-2317(24)00173-3. [Epub ahead of print]73 103195

Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice.

Ye Chen, Jiaqi Zhang, Ying Tian, Xiangning Xu, Bicheng Wang, Ziqi Huang, Shuo Lou, Jingyi Kang, Ningning Zhang, Jing Weng, Yuanjing Liang, Wei Ma.

Accumulating oxidative damage is a primary driver of ovarian reserve decline along with aging. However, the mechanism behind the imbalance in reactive oxygen species (ROS) is not yet fully understood. Here we investigated changes in iron metabolism and its relationship with ROS disorder in aging ovaries of mice. We found increased iron content in aging ovaries and oocytes, along with abnormal expression of iron metabolic proteins, including heme oxygenase 1 (HO-1), ferritin heavy chain (FTH), ferritin light chain (FTL), mitochondrial ferritin (FTMT), divalent metal transporter 1 (DMT1), ferroportin1(FPN1), iron regulatory proteins (IRP1 and IRP2) and transferrin receptor 1 (TFR1). Notably, aging oocytes exhibited enhanced ferritinophagy and mitophagy, and consistently, there was an increase in cytosolic Fe2+, elevated lipid peroxidation, mitochondrial dysfunction, and augmented lysosome activity. Additionally, the ovarian expression of p53, p21, p16 and microtubule-associated protein tau (Tau) were also found to be upregulated. These alterations could be phenocopied with in vitro Fe2+ administration in oocytes from 2-month-old mice but were alleviated by deferoxamine (DFO). In vivo application of DFO improved ovarian iron metabolism and redox status in 12-month-old mice, and corrected the alterations in cytosolic Fe2+, ferritinophagy and mitophagy, as well as related degenerative changes in oocytes. Thereby in the whole, DFO delayed the decline in ovarian reserve and significantly increased the number of superovulated oocytes with reduced fragmentation and aneuploidy. Together, our findings suggest that aging-related disturbance in ovarian iron homeostasis contributes to excessive ROS production and that iron chelation may improve ovarian redox status, and efficiently delay the decline in ovarian reserve and oocyte quality in aging mice. These data propose a novel intervention strategy for preserving the ovarian reserve function in elderly women.

DOI: https://doi.org/10.1016/j.redox.2024.103195
Science. 2024 May 23. eadk4898

A metabolic dependency of EBV can be targeted to hinder B cell transformation.

Swiss Transplant Cohort Study#

Following infection of B cells, Epstein Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates NAD de novo biosynthesis by driving expression of the metabolic enzyme IDO1 in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match ATP-production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is, thus, a druggable metabolic vulnerability of EBV-driven B cell transformation-opening therapeutic possibilities for EBV-related diseases.

DOI: https://doi.org/10.1126/science.adk4898
Nat Commun. 2024 May 18. 15(1): 4247

Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys.

Alexandra S Piotrowski-Daspit, Laura G Bracaglia, David A Eaton, Owen Richfield, Thomas C Binns, Claire Albert, Jared Gould, Ryland D Mortlock, Marie E Egan, Jordan S Pober, W Mark Saltzman.

The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs-and perhaps other NPs-can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.

DOI: https://doi.org/10.1038/s41467-024-48442-7
Metabolites. 2024 Apr 25. pii: 246. [Epub ahead of print]14(5):

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Nils W F Meijer, Susan Zwakenberg, Johan Gerrits, Denise Westland, Arif I Ardisasmita, Sabine A Fuchs, Nanda M Verhoeven-Duif, Judith J M Jans, Fried J T Zwartkruis.

  Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

Keywords:  TCA cycle; direct infusion–high-resolution mass spectrometry; glutaminolysis; glycolysis; isotope tracing; organoids; patient material

DOI:  https://doi.org/10.3390/metabo14050246
Nat Commun. 2024 May 18. 15(1): 4239

Preserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG.

Edoardo José Longarini, Ivan Matić.

Ester-linked post-translational modifications, including serine and threonine ubiquitination, have gained recognition as important cellular signals. However, their detection remains a significant challenge due to the chemical lability of the ester bond. This is the case even for long-known modifications, such as ADP-ribosylation on aspartate and glutamate, whose role in PARP1 signaling has recently been questioned. Here, we present easily implementable methods for preserving ester-linked modifications. When combined with a specific and sensitive modular antibody and mass spectrometry, these approaches reveal DNA damage-induced aspartate/glutamate mono-ADP-ribosylation. This previously elusive signal represents an initial wave of PARP1 signaling, contrasting with the more enduring nature of serine mono-ADP-ribosylation. Unexpectedly, we show that the poly-ADP-ribose hydrolase PARG is capable of reversing ester-linked mono-ADP-ribosylation in cells. Our methodology enables broad investigations of various ADP-ribosylation writers and, as illustrated here for noncanonical ubiquitination, it paves the way for exploring other emerging ester-linked modifications.

DOI: https://doi.org/10.1038/s41467-024-48314-0
Nat Commun. 2024 May 20. 15(1): 4276

Production of deoxycholic acid by low-abundant microbial species is associated with impaired glucose metabolism.

Annika Wahlström, Ariel Brumbaugh, Wilhelm Sjöland, Lisa Olsson, Hao Wu, Marcus Henricsson, Annika Lundqvist, Kassem Makki, Stanley L Hazen, Göran Bergström, Hanns-Ulrich Marschall, Michael A Fischbach, Fredrik Bäckhed.

Alterations in gut microbiota composition are suggested to contribute to cardiometabolic diseases, in part by producing bioactive molecules. Some of the metabolites are produced by very low abundant bacterial taxa, which largely have been neglected due to limits of detection. However, the concentration of microbially produced metabolites from these taxa can still reach high levels and have substantial impact on host physiology. To explore this concept, we focused on the generation of secondary bile acids by 7α-dehydroxylating bacteria and demonstrated that addition of a very low abundant bacteria to a community can change the metabolic output dramatically. We show that Clostridium scindens converts cholic acid into the secondary bile acid deoxycholic acid (DCA) very efficiently even though the abundance of C. scindens is low, but still detectable by digital droplet PCR. We also show that colonization of germ-free female mice with a community containing C. scindens induces DCA production and affects host metabolism. Finally, we show that DCA correlates with impaired glucose metabolism and a worsened lipid profile in individuals with type 2 diabetes, which implies that this metabolic pathway may contribute to the development of cardiometabolic disease.

DOI: https://doi.org/10.1038/s41467-024-48543-3
Nat Med. 2024 May 21.

A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.

Lisa Kinget, Stefan Naulaerts, Jannes Govaerts, Isaure Vanmeerbeek, Jenny Sprooten, Raquel S Laureano, Nikolina Dubroja, Gautam Shankar, Francesca M Bosisio, Eduard Roussel, Annelies Verbiest, Francesca Finotello, Markus Ausserhofer, Diether Lambrechts, Bram Boeckx, Agnieszka Wozniak, Louis Boon, Johan Kerkhofs, Jessica Zucman-Rossi, Maarten Albersen, Marcella Baldewijns, Benoit Beuselinck, Abhishek D Garg.

An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.

DOI: https://doi.org/10.1038/s41591-024-02978-9
Cell Mol Life Sci. 2024 May 22. 81(1): 227

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Lisa Gallwitz, Florian Bleibaum, Matthias Voss, Michaela Schweizer, Katharina Spengler, Dominic Winter, Frederic Zöphel, Stephan Müller, Stefan Lichtenthaler, Markus Damme, Paul Saftig.

  Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Keywords:  Amyloid precursor protein; Autophagy; Bulk proteolysis; Cathepsins; Lysosome; Protease network; Proteolysis; Proteomics

DOI:  https://doi.org/10.1007/s00018-024-05274-4
Front Physiol. 2024 ;15 1352242

High nitrate levels in skeletal muscle contribute to nitric oxide generation via a nitrate/nitrite reductive pathway in mice that lack the nNOS enzyme.

Supranee Upanan, Jeeyoung Lee, Khalid J Tunau-Spencer, Praveen K Rajvanshi, Elizabeth C Wright, Constance T Noguchi, Alan N Schechter.

  Introduction: Nitric oxide (NO) is a vasodilator gas that plays a critical role in mitochondrial respiration and skeletal muscle function. NO is endogenously generated by NO synthases: neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), or inducible NO synthase (iNOS). NO in skeletal muscle is partly generated by nNOS, and nNOS deficiency can contribute to muscular dystrophic diseases. However, we and others discovered an alternative nitrate/nitrite reductive pathway for NO generation: nitrate to nitrite to NO. We hypothesized that nitrate supplementation would increase nitrate accumulation in skeletal muscle and promote a nitrate/nitrite reductive pathway for NO production to compensate for the loss of nNOS in skeletal muscle.Methods: Wild-type (WT) and genetic nNOS knockout (nNOS-/-) mice were fed normal chow (386.9 nmol/g nitrate) and subjected to three treatments: high-nitrate water (1 g/L sodium nitrate for 7 days), low-nitrate diet (46.8 nmol/g nitrate for 7 days), and low-nitrate diet followed by high-nitrate water for 7 days each.
Results: High-nitrate water supplementation exhibited a greater and more significant increase in nitrate levels in skeletal muscle and blood in nNOS-/- mice than in WT mice. A low-nitrate diet decreased blood nitrate and nitrite levels in both WT and nNOS-/- mice. WT and nNOS-/- mice, treated with low-nitrate diet, followed by high-nitrate water supplementation, showed a significant increase in nitrate levels in skeletal muscle and blood, analogous to the increases observed in nNOS-/- mice supplemented with high-nitrate water. In skeletal muscle of nNOS-/- mice on high-nitrate water supplementation, on low-nitrate diet, and in low-high nitrate treatment, the loss of nNOS resulted in a corresponding increase in the expression of nitrate/nitrite reductive pathway-associated nitrate transporters [sialin and chloride channel 1 (CLC1)] and nitrate/nitrite reductase [xanthine oxidoreductase (XOR)] but did not show a compensatory increase in iNOS or eNOS protein and eNOS activation activity [p-eNOS (Ser1177)].
Discussion: These findings suggest that a greater increase in nitrate levels in skeletal muscle of nNOS-/- mice on nitrate supplementation results from reductive processes to increase NO production with the loss of nNOS in skeletal muscle.

Keywords:  neuronal nitric oxide synthase; neuronal nitric oxide synthase knockout mice (nNOS−/−); nitrate; nitrate/nitrite reductive pathway; nitric oxide; nitric oxide synthases; skeletal muscle; xanthine oxidoreductase

DOI:  https://doi.org/10.3389/fphys.2024.1352242
Gut. 2024 May 22. pii: gutjnl-2023-331447. [Epub ahead of print]

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis.

Mrigya Babuta, Caroline Morel, Marcelle de Carvalho Ribeiro, Charles Calenda, Martí Ortega-Ribera, Prashanth Thevkar Nagesh, Christopher Copeland, Yuan Zhuang, Yanbo Wang, Yeonhee Cho, Radhika Joshi, Viliam Brezani, Danielle Hawryluk, Aditi Ashish Datta, Jeeval Mehta, Imad Nasser, Gyongyi Szabo.

  OBJECTIVE: Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH.DESIGN: C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated.
RESULTS: We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract).
CONCLUSION: Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.

Keywords:  ALCOHOLIC LIVER DISEASE; FIBROSIS; HEPATIC STELLATE CELL; MACROPHAGES

DOI:  https://doi.org/10.1136/gutjnl-2023-331447