bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2022‒12‒18
25 papers selected by
Erika Mariana Palmieri
NIH/NCI Laboratory of Cancer ImmunoMetabolism

  1. JCI Insight. 2022 11 22. pii: e138539. [Epub ahead of print]7(22):
      Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein's self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88‑dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr-/- mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88‑dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.
    Keywords:  Inflammation; Innate immunity; Vascular Biology
  2. Sci Adv. 2022 Dec 16. 8(50): eabp8293
      Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase.
  3. Cell Metab. 2022 Dec 08. pii: S1550-4131(22)00502-2. [Epub ahead of print]
      The mammalian succinate dehydrogenase (SDH) complex has recently been shown as capable of operating bidirectionally. Here, we develop a method (Q-Flux) capable of measuring absolute rates of both forward (VSDH(F)) and reverse (VSDH(R)) flux through SDH in vivo while also deconvoluting the amount of glucose derived from four discreet carbon sources in the liver. In validation studies, a mitochondrial uncoupler increased net SDH flux by >100% in awake rodents but also increased SDH cycling. During hyperglucagonemia, attenuated pyruvate cycling enhances phosphoenolpyruvate carboxykinase efficiency to drive increased gluconeogenesis, which is complemented by increased glutaminase (GLS) flux, methylmalonyl-CoA mutase (MUT) flux, and glycerol conversion to glucose. During hyperinsulinemic-euglycemic clamp, both pyruvate carboxylase and GLS are suppressed, while VSDH(R) is increased. Unstimulated MUT is a minor anaplerotic reaction but is readily induced by small amounts of propionate, which elicits glucagon-like metabolic rewiring. Taken together, Q-Flux yields a comprehensive picture of hepatic mitochondrial metabolism and should be broadly useful to researchers.
    Keywords:  anaplerosis; glucagon; glutaminase; insulin; mass spectrometry; metabolic flux analysis; methylmalonyl-CoA mutase; mitochondrial metabolism; propionate; succinate dehydrogenase
  4. Commun Biol. 2022 Dec 13. 5(1): 1366
      Cellular metabolism influences immune cell function, with mitochondrial fatty acid β-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection. Screening the Cpt1a gene for common variants predicted to affect protein function revealed allele rs2229738_T, which was associated with pneumonia risk in a targeted human phenome association study. Pharmacologic inhibition of Cpt1a increases mortality and impairs control of the infection in a murine model of bacterial pneumonia. Susceptibility to pneumonia is associated with blunted neutrophilic responses in mice and humans that result from impaired neutrophil trafficking to the site of infection. Chemotaxis responsible for neutrophil trafficking requires Cpt1a-dependent mitochondrial fatty acid oxidation for amplification of chemoattractant signals. These findings identify Cpt1a as a potential host determinant of infection susceptibility and demonstrate a requirement for mitochondrial fatty acid oxidation in neutrophil biology.
  5. Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01697-7. [Epub ahead of print]41(11): 111809
      The gut microbiota influences acetylation on host histones by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-coenzyme A (CoA), a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.
    Keywords:  CP: Microbiology; colitis; epigenetics; fatty acid metabolism; histone acetylation; host-microbiota interactions
  6. Immunity. 2022 Dec 09. pii: S1074-7613(22)00603-3. [Epub ahead of print]
      Obesity-induced chronic liver inflammation is a hallmark of nonalcoholic steatohepatitis (NASH)-an aggressive form of nonalcoholic fatty liver disease. However, it remains unclear how such a low-grade, yet persistent, inflammation is sustained in the liver. Here, we show that the macrophage phagocytic receptor TREM2, induced by hepatocyte-derived sphingosine-1-phosphate, was required for efferocytosis of lipid-laden apoptotic hepatocytes and thereby maintained liver immune homeostasis. However, prolonged hypernutrition led to the production of proinflammatory cytokines TNF and IL-1β in the liver to induce TREM2 shedding through ADAM17-dependent proteolytic cleavage. Loss of TREM2 resulted in aberrant accumulation of dying hepatocytes, thereby further augmenting proinflammatory cytokine production. This ultimately precipitated a vicious cycle that licensed chronic inflammation to drive simple steatosis transition to NASH. Therefore, impaired macrophage efferocytosis is a previously unrecognized key pathogenic event that enables chronic liver inflammation in obesity. Blocking TREM2 cleavage to restore efferocytosis may represent an effective strategy to treat NASH.
    Keywords:  TREM2; chronic inflammation; efferocytosis; nonalcoholic steatohepatitis; proinflammatory cytokines
  7. Blood Adv. 2022 Dec 15. pii: bloodadvances.2022008272. [Epub ahead of print]
      Cancer cells take up amino acids from the extracellular space to drive cell proliferation and viability. Similar mechanisms are employed by immune cells. The result is competition between conventional T cells, or indeed CAR-T cells, and tumour cells for limited availability of amino acids within the environment. We demonstrate that T cells can be re-engineered to express SLC7A5 or SLC7A11 transmembrane amino acid transporters alongside chimeric antigen receptors (CAR). Transporter modifications increase CAR-T cell proliferation under low tryptophan or cystine conditions with no loss of CAR cytotoxicity or increased exhaustion. Transcriptomic and phenotypic analysis reveals that downstream, SLC7A5/SLC7A11 modified CAR-T cells upregulate intracellular Arginase expression and activity. In turn we engineer and phenotype a further generation of CAR-T cells which express functional Arginase I/Arginase II enzymes, and have enhanced CAR-T cell proliferation and anti-tumour activity. Thus CAR-T cells can be adapted to the amino acid metabolic microenvironment of cancer, a hitherto recognised but unaddressed barrier to successful CAR-T therapy.
  8. Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01710-7. [Epub ahead of print]41(11): 111818
      Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that in vitro and in vivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.
    Keywords:  CP: Cancer; Drp1; ETC; KRas; NAD; Opa1; cancer; cristae; fission; fusion; mitochondria
  9. Mol Cell. 2022 Dec 08. pii: S1097-2765(22)01130-3. [Epub ahead of print]
      Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.
    Keywords:  cell cycle; macrophage plasticity; macrophage polarization; single-cell epigenomics
  10. Cell Chem Biol. 2022 Dec 01. pii: S2451-9456(22)00415-9. [Epub ahead of print]
      While it is well known that expression levels of metabolic enzymes regulate the metabolic state of the cell, there is mounting evidence that the converse is also true, that metabolite levels themselves can modulate gene expression via epigenetic modifications and transcriptional regulation. Here we focus on the one-carbon metabolic pathway, which provides the essential building blocks of many classes of biomolecules, including purine nucleotides, thymidylate, serine, and methionine. We review the epigenetic roles of one-carbon metabolic enzymes and their associated metabolites and introduce an interactive computational resource that places enzyme essentiality in the context of metabolic pathway topology. Therefore, we briefly discuss examples of metabolic condensates and higher-order complexes of metabolic enzymes downstream of one-carbon metabolism. We speculate that they may be required to the formation of transcriptional condensates and gene expression control. Finally, we discuss new ways to exploit metabolic pathway compartmentalization to selectively target these enzymes in cancer.
    Keywords:  cancer; chromatin; epigenetics; folate metabolism; metabolic condensates; nuclear condensates; nuclear metabolism; nucleotides; one-carbon metabolism; phase separation; purinergic signaling; transcription regulation; transcriptional condensates
  11. Cancer Res. 2022 Dec 16. pii: CAN-22-1837. [Epub ahead of print]
      Immunotherapy can elicit robust anticancer responses in the clinic. However, a large proportion of patients with colorectal cancer (CRC) do not benefit from treatment. While previous studies have shown that hydrogen sulfide (H2S) is involved in CRC development and immune escape, further insights into the mechanisms and related molecules are needed to identify approaches to reverse the tumor supportive functions of H2S. Here, we observed significantly increased H2S levels in CRC tissues. Decreasing H2S levels by using CBS+/- mice or feeding mice a sulfur amino acid-restricted diet (SARD) led to a marked decrease in differentiated CD4+CD25+Foxp3+ Tregs and an increase in the CD8+ T cell/Treg ratio. Endogenous or exogenous H2S depletion enhanced the efficacy of anti-PD-L1 and anti-CTLA-4 treatment. H2S promoted Treg activation through persulfidation of ENO1 at cysteine 119. Furthermore, H2S inhibited the migration of CD8+ T cells by increasing the expression of AAK-1 via ELK4 persulfidation at cysteine 25. Overall, reducing H2S levels engenders a favorable immune microenvironment in CRC by decreasing the persulfidation of ENO1 in Tregs and ELK4 in CD8+ T cells. SARD represents a potential dietary approach to promote responses to immunotherapies in CRC.
  12. Science. 2022 Dec 16. 378(6625): eabq5209
      Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.
  13. STAR Protoc. 2022 Dec 15. pii: S2666-1667(22)00825-5. [Epub ahead of print]4(1): 101945
      The lipid peroxidation level of tumor-infiltrating CD8+ T cells is crucial for its activity and longevity. Here, we describe a protocol for effective and epitope-preserving dissociation of mouse tumors and subsequent leukocyte purification and lipid peroxidation staining of adoptively transferred CD8+ T cells. We use BODIPY 581/591 C11 to monitor the cellular lipid peroxidation level and detect its fluorescent change by flow cytometry, followed by analysis in FlowJo. This protocol is adaptable to intrinsic CD8+ T cells in tumors as well. For complete details on the use and execution of this protocol, please refer to Xiao et al. (2022)1 and Ma et al. (2021).2.
    Keywords:  Cancer; Cell Biology; Flow Cytometry/Mass Cytometry; Immunology; Metabolism; Molecular/Chemical Probes; Single Cell
  14. Proc Natl Acad Sci U S A. 2022 Dec 20. 119(51): e2212879119
      Epithelial-to-mesenchymal transition (EMT) is a dramatic change in cellular physiology during development and metastasis, which requires coordination between cell signaling, adhesion, and membrane protrusions. These processes all involve dynamic changes in the plasma membrane; yet, how membrane lipid content regulates membrane function during EMT remains incompletely understood. By screening for differential expression of lipid-modifying genes over the course of EMT in the avian neural crest, we have identified the ceramide-producing enzyme neutral sphingomyelinase 2 (nSMase2) as a critical regulator of a developmental EMT. nSMase2 expression begins at the onset of EMT, and in vivo knockdown experiments demonstrate that nSMase2 is necessary for neural crest migration. We find that nSMase2 promotes Wnt and BMP signaling and is required to activate the mesenchymal gene expression program. Mechanistically, we show that nSMase2-dependent ceramide production is necessary for and sufficient to up-regulate endocytosis and is required for Wnt co-receptor internalization. Finally, inhibition of endocytosis in the neural crest mimics the loss of migration and Wnt signaling observed following nSMase2 knockdown. Our results support a model in which nSMase2 is expressed at the onset of neural crest EMT to produce ceramide and facilitate receptor-mediated endocytosis of Wnt and BMP signaling complexes, thereby activating promigratory gene expression. These results highlight the critical role of plasma membrane lipid metabolism in regulating transcriptional changes during developmental EMT programs.
    Keywords:  cell signaling; ceramide; endocytosis; epithelial-to-mesenchymal transition; neural crest
  15. J Clin Invest. 2022 Dec 13. pii: e164508. [Epub ahead of print]
      Disuse osteoporosis is a metabolic bone disease resulted from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during re-ambulation remains an unresolved medical challenge. Here, we demonstrated that loading-induced increase in bone architecture/strength was suppressed in skeletons previously exposed to unloading. This reduction in bone mechanosensitivity was directly associated with attenuated osteocytic Ca2+ oscillatory dynamics. The unloading-induced compromised osteocytic Ca2+ response to reloading resulted from the HIF-1α/PDK1 axis-mediated increase in glycolysis, and a subsequent reduction in ATP synthesis. HIF-1α also transcriptionally induced substantial glutaminase 2 expression and thereby glutamine addiction in osteocytes. Inhibition of glycolysis by blocking PDK1 or glutamine supplementation restored the mechanosensitivity in those skeletons with previous unloading by fueling the tricarboxylic acid cycle and rescuing subsequent Ca2+ oscillations in osteocytes. Thus, we provide a mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.
    Keywords:  Bioenergetics; Bone Biology; Bone disease; Calcium signaling
  16. Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01674-6. [Epub ahead of print]41(11): 111786
      24 h whole-body substrate metabolism and the circadian clock within skeletal muscle are both compromised upon metabolic disease in humans. Here, we assessed the 24 h muscle metabolome by serial muscle sampling performed under 24 h real-life conditions in young, healthy (YH) men versus older, metabolically compromised (OMC) men. We find that metabolites associated with the initial steps of glycolysis and hexosamine biosynthesis are higher in OMC men around the clock, whereas metabolites associated with glutamine-alpha-ketoglutarate, ketone, and redox metabolism are lower in OMC men. The night period shows the largest number of differently expressed metabolites. Both groups demonstrate 24 h rhythmicity in half of the metabolome, but rhythmic metabolites only partially overlap. Specific metabolites are only rhythmic in YH men (adenosine), phase shifted in OMC men (cis-aconitate, flavin adenine dinucleotide [FAD], and uridine diphosphate [UDP]), or have a reduced 24 h amplitude in OMC men (hydroxybutyrate and hippuric acid). Our data highlight the plasticity of the skeletal muscle metabolome over 24 h and large divergence across the metabolic health spectrum.
    Keywords:  CP: Metabolism; FAD; PGK1; adenosine; circadian rhythm; glycolysis; hexosamine; hydroxybutyrate; insulin resistance; metabolomics; misalignment
  17. Clin Transl Oncol. 2022 Dec 15.
      BACKGROUND: Adipose tissue is a major component of breast stroma. This study focused on delineating the effects of adipose stem cells (ASCs) derived from breast of healthy women and cancer patients with normal or tumor breast cells.METHODS: The ASCs were induced to differentiate into adipocytes, and the subsequent adipocyte conditioned media (ACM) were evaluated for their fatty acid profile, adipokine secretion and influence on proliferation, migration and invasion on tumoral (MCF-7 and SUM159) and normal (HMEC) human breast cell lines.
    RESULTS: An enrichment of arachidonic acid was observed in ACM from tumor tissues. Adipose tissues from tumor free secrete twice as much leptin than those from proximal or distal to the tumor. All ACMs display proliferative activity and favor invasiveness of SUM159 cells compared to MCF-7 and HMEC. All ACMs induced lipid droplets accumulation in MCF-7 cells and increased CD36 expression in tumor cells.
    CONCLUSION: We conclude that among secreted factors analyzed, only arachidonic acid and leptin levels did discriminate ASCs from tumor-bearing and tumor-free breasts emphasizing the importance that other cell types could contribute to the adipose tissue secretome in a tumor context.
    Keywords:  Adipose stem cells; Breast cancer; Breast-associated adipocytes; Microenvironment
  18. Sci Immunol. 2022 Dec 23. 7(78): eade5728
      Interleukin-1 (IL-1) family cytokines are key barrier cytokines that are typically expressed as inactive, or partially active, precursors that require proteolysis within their amino termini for activation. IL-37 is an enigmatic member of the IL-1 family that has been proposed to be activated by caspase-1 and to exert anti-inflammatory activity through engagement of the IL-18R and SIGIRR. However, here we show that the longest IL-37 isoform, IL-37b, exhibits robust proinflammatory activity upon amino-terminal proteolysis by neutrophil elastase or cathepsin S. In sharp contrast, caspase-1 failed to process or activate IL-37 at concentrations that robustly activated its canonical substrate, IL-1β. IL-37 and IL-36 exhibit high structural homology, and, consistent with this, a K53-truncated form of IL-37, mimicking the cathepsin S-processed form of this cytokine, was found to exert its proinflammatory effects via IL-36 receptor engagement and produced an inflammatory signature practically identical to IL-36. Administration of K53-truncated IL-37b intraperitoneally into wild-type mice also elicited an inflammatory response that was attenuated in IL-36R-/- animals. These data demonstrate that, in common with other IL-1 family members, mature IL-37 can also elicit proinflammatory effects upon processing by specific proteases.
  19. Redox Biol. 2022 Dec 01. pii: S2213-2317(22)00331-7. [Epub ahead of print]59 102559
      Alcoholic liver disease (ALD) is associated with hepatic inflammatory activation and iron overload. The receptor for advanced glycation end products (RAGE) is an important metabolic mediator during the development of ALD. The aim of this study was to determine the effect of RAGE on iron homeostasis in ALD. We found increased circulating transferrin, hepcidin and ferritin in ALD patients and positively correlated with RAGE level. RAGE knockout (RAGE-/-) and wild-type mice were subjected to chronic alcoholic feeding for 6 weeks to induce ALD, and RAGE inhibitor, iron chelator or lipid peroxidation inhibitor were administered. We showed that chronic alcohol administration triggered hepatic steatosis, inflammation, and oxidative stress, which were eliminated by deficiency or inhibition of RAGE. Surprisingly, pathways of hepatic iron metabolism were significantly altered, including increased iron uptake (Tf/TfR) and storage (Ferritin), as well as decreased iron export (FPN1/Hepcidin). In vitro experiments confirmed that RAGE had different effects on the mechanism of iron metabolism of hepatocytes and macrophages respectively. In conclusion, our data revealed preclinical evidence for RAGE inhibition as an effective intervention for alleviating alcohol-induced liver injury.
    Keywords:  Inflammation; Iron metabolism; Lipid peroxidation; Macrophages; Oxidative stress; RAGE
  20. Cells. 2022 Nov 23. pii: 3738. [Epub ahead of print]11(23):
      Mesenchymal stromal cells (MSCs) represent a promising treatment for immune-related diseases due to their diverse immunomodulatory paracrine functions. However, progress of culture-expanded MSCs is hindered by inconsistent cell function, poor localization, and insufficient retention when administered as suspended cell injections, thus placing spatiotemporal dosing constraints on therapeutic functions. To address these limitations, we introduce the combination of in vitro interferon-gamma (IFN-γ) priming, a key stimulator of MSC immunosuppressive potency, and thermoresponsive cultureware to harvest cultured MSCs as directly transplantable scaffold-free immunosuppressive cell sheets. Here, we demonstrate that MSC sheets produced with IFN-γ priming upregulate expression of immunosuppressive factors indoleamine 2,3-dioxygenase (IDO-1), interleukin-10 (IL-10), programmed death ligand-1 (PD-L1), and prostaglandin E2 (PGE2) in both dose- and duration-dependent manners. In addition, IFN-γ primed MSC sheets showed increased ability to inhibit T-cell proliferation via indirect and direct contact, specifically related to increased IDO-1 and PGE2 concentrations. Furthermore, this study's use of human clinical-grade single-cell-derived clonal bone marrow-derived MSCs, contributes to the future translatability and clinical relevancy of the produced sheets. Ultimately, these results present the combination of IFN-γ priming and MSC sheets as a new strategy to improve MSC-mediated treatment of localized inflammatory diseases.
    Keywords:  cellular therapy; coculture; immunomodulation; licensing; mesenchymal stem cells; pre-conditioning; tissue engineering
  21. Nat Cardiovasc Res. 2022 Nov;1 1056-1071
      Dissecting the pathways regulating the adaptive immune response in atherosclerosis is of particular therapeutic interest. Here we report that the lipid G-protein coupled receptor GPR55 is highly expressed by splenic plasma cells (PC), upregulated in mouse spleens during atherogenesis and human unstable or ruptured compared to stable plaques. Gpr55-deficient mice developed larger atherosclerotic plaques with increased necrotic core size compared to their corresponding controls. Lack of GPR55 hyperactivated B cells, disturbed PC maturation and resulted in immunoglobulin (Ig)G overproduction. B cell-specific Gpr55 depletion or adoptive transfer of Gpr55-deficient B cells was sufficient to promote plaque development and elevated IgG titers. In vitro, the endogenous GPR55 ligand lysophsophatidylinositol (LPI) enhanced PC proliferation, whereas GPR55 antagonism blocked PC maturation and increased their mitochondrial content. Collectively, these discoveries provide previously undefined evidence for GPR55 in B cells as a key modulator of the adaptive immune response in atherosclerosis.
  22. J Transl Med. 2022 Dec 12. 20(1): 581
      BACKGROUND: The poor prognosis of ovarian cancer patients is strongly related to peritoneal metastasis with the production of malignant ascites. However, it remains largely unclear how ascites in the peritoneal cavity influences tumor metabolism and recurrence. This study is an explorative approach aimed at for a deeper molecular and physical-chemical characterization of malignant ascites and to investigate their effect on in vitro ovarian cancer cell proliferation.METHODS: This study included 10 malignant ascites specimens from patients undergoing ovarian cancer resection. Ascites samples were deeply phenotyped by 1H-NMR based metabolomics, blood-gas analyzer based gas flow analysis and flow cytomertry based a 13-plex cytokine panel. Characteristics of tumor cells were investigated in a 3D spheroid model by SEM and metabolic activity, adhesion, anti-apoptosis, migratory ability evaluated by MTT assay, adhesion assay, flowcytometry and scratch assay. The effect of different pH values was assessed by adding 10% malignant ascites to the test samples.
    RESULTS:  The overall extracellular (peritoneal) environment was alkaline, with pH of ascites at stage II-III = 7.51 ± 0.16, and stage IV = 7.78 ± 0.16. Ovarian cancer spheroids grew rapidly in a slightly alkaline environment. Decreasing pH of the cell culture medium suppressed tumor features, metabolic activity, adhesion, anti-apoptosis, and migratory ability. However, 10% ascites could prevent tumor cells from being affected by acidic pH. Metabolomics analysis identified stage IV patients had significantly higher concentrations of alanine, isoleucine, phenylalanine, and glutamine than stage II-III patients, while stage II-III patients had significantly higher concentrations of 3-hydroxybutyrate. pH was positively correlated with acetate, and acetate positively correlated with lipid compounds. IL-8 was positively correlated with lipid metabolites and acetate. Glutathione and carnitine were negatively correlated with cytokines IL-6 and chemokines (IL-8 & MCP-1).
    CONCLUSION: Alkaline malignant ascites facilitated ovarian cancer progression. Additionally, deep ascites phenotyping by metabolomics and cytokine investigations allows for a refined stratification of ovarian cancer patients. These findings contribute to the understanding of ascites pathology in ovarian cancer.
    Keywords:  Cell culture; Cytokine; In vitro; In vivo; Metabolic profile; Peritoneal fluid; pH
  23. Mitochondrion. 2022 Dec 11. pii: S1567-7249(22)00108-8. [Epub ahead of print]
      While ketone bodies support overall brain energy metabolism, it is increasingly clear specific brain cell types respond differently to ketone body availability. Here, we characterized how SH-SY5Y neuroblastoma cell, primary neuron, and primary astrocyte bioenergetics and nutrient sensing pathways respond to β-hydroxybutyrate (βOHB). SH-SY5Y cells and primary neurons, but not astrocytes, exposed to βOHB increased respiration and decreased PI3K-Akt-mTOR signaling. Despite increased carbon availability and respiration, SH-SY5Y cells treated with βOHB reduced their overall metabolic activity and cell cycling rate. Levels of the quiescence-regulating Yamanaka factors increased to a broader extent in SH-SY5Y cells and primary neurons. We propose a βOHB-induced increase in neuron respiration, accompanied by activation of quiescence associated pathways, could alleviate bioenergetic stress and limit cell senescence. This in turn could potentially benefit conditions, including brain aging and neurodegenerative diseases, that feature bioenergetic decline and cell senescence.
    Keywords:  Ketones; aging; bioenergetics; mitochondria; quiescence; senescence
  24. J Biol Chem. 2022 Dec 09. pii: S0021-9258(22)01236-4. [Epub ahead of print] 102793
      Astrocytic excitatory amino acid transporter 2 (EAAT2) plays a major role in removing the excitatory neurotransmitter L-glutamate (L-Glu) from synaptic clefts in the forebrain to prevent excitotoxicity. Polyunsaturated fatty acids such as docosahexaenoic acid (DHA, 22:6 n-3) enhance synaptic transmission, and their target molecules include EAATs. Here, we aimed to investigate the effect of DHA on EAAT2 and identify the key amino acid for DHA/EAAT2 interaction by electrophysiological recording of L-Glu-induced current in Xenopus oocytes transfected with EAATs, their chimeras, and single mutants. DHA transiently increased the amplitude of EAAT2, but tended to decrease that of EAAT1, another astrocytic EAAT. Single mutation of leucine (Leu) 434 to alanine (Ala) completely suppressed the augmentation by DHA, while mutation of EAAT1 Ala 435 (corresponding to EAAT2 Leu434) to Leu changed the effect from suppression to augmentation. Other polyunsaturated fatty acids (PUFAs) (docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid, and a-linolenic acid) similarly augmented the EAAT2 current and suppressed the EAAT1 current. Finally, our docking analysis suggested the most stable docking site is the lipid crevice of EAAT2, in close proximity to the L-Glu and sodium binding sites, suggesting that the DHA/Leu434 interaction might affect the elevator-like slide and/or the shapes of the other binding sites. Collectively, our results highlight a key molecular detail in the DHA-induced regulation of synaptic transmission involving EAATs.
    Keywords:  astrocyte; electrophysiology; glutamate; polyunsaturated fatty acid (PUFA); transporter
  25. FEBS Open Bio. 2022 Dec 15.
      Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane-bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy-based approaches to investigate Mtb responses to acidic environments in cellulo. We report high-content imaging modalities to probe Mtb sensing of external pH or visualise in real-time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step-by-step analyses that enable robust image-based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb-host cell interactions. These methods can be adapted to study host-pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival.
    Keywords:  Tuberculosis; bacterial reporters; endolysosomes; high content fluorescence microscopy; human induced pluripotent stem cells-derived macrophages; pH sensing and homeostasis