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Relationship between sterol synthesis and DNA synthesis in phytohem-agglutin-stimulated mouse lymphocytes
Abstract
Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. Rates of incorporation of radioactivity from [14-C]acetate into fatty acids and into CO2 by phytohemagglutinin-treated blood were not significantly different from control values. Phytohemagglutinin stimulation of sterol synthesis could be abolished by the addition of certain oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol and 20alpha-hydroxycholesterol) which specifically depress the activity of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl CoA reductase [mevalonate:NADP-nOXIDOREDUCTASE (CoA acylating); EC 1.1.1.34]. This treatment also abolished DNA synthesis and blastogenesis which otherwise followed the peak of sterol synthesis. Furthermore, DNA synthesis was repressed only if the inhibitor was added early enough to prevent sterol synthesis from reaching its usual maximum. When the compound was added after the rate of sterol synthesis had reached its maximum, DNA synthesis was not affected. These findings suggest that the synthesis of cholesterol is an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes after phytohemagglutinin activation.
... Since IL-27-stimulated T cells were identified as a source of 25OHC, we next sort to determine how secreted 25OHC might influence the cellular phenotype of the surrounding T cells via suppressing cholesterol synthesis. According to previous reports, 25OHC mediated suppression of sterol synthesis, abolished DNA synthesis and lymphocyte proliferation (25)(26)(27)(28). 25OHC was exogenously added to the in vitro culture of T cells stimulated with TCR, and cell division was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling. ...
... 25OHC was exogenously added to the in vitro culture of T cells stimulated with TCR, and cell division was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling. Among the doses of 25OHC tested, cell proliferation was significantly suppressed at 100 nM, and almost completely abrogated at 1000 nM ( Fig. 4A), in line with previous reports (26)(27)(28). When cell viability was evaluated by a combination of 7aminoactinomycin D (7-ADD) staining and cell size (FSC), impaired viability was observed in a dose-dependent manner (Fig. 4B). ...
... Removal of IL-2 signaling resulted in slowed cell growth, and the reduction in the proliferative state effectively saved cells from cell death otherwise caused by 25OHC in vitro. Our pathway analysis of 25OHC-treated cells also revealed the cell cycle, DNA repair, and centromeres as the biological processes affected by high concentrations of 25OHC in line with previous reports describing suppressive effect of 25OHC on activated lymphocyte proliferation (26)(27)(28). Since notable concentrations of 25OHC (~ 300 nM) were detected in the serum of mice administered the TLR4 agonist (42), it is reasonable to believe that states of immune cell activation and proliferation, rather than effector phenotypes such as Th1 and Th17 e.g., are key factors that determine whether a particular subset of immune cells is or is not affected by 25OHC. ...
Interleukin-27 (IL-27) is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4⁺ T cells by IL-27, enhanced by transforming growth factor–β (TGF-β), and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient deprivation cell death without releasing high-mobility group box 1 (HMGB1). This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Ch25h-expressing CD4⁺ T cells that received IL-27 and TGF-β signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27–treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worse outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.
... Besides this well described reprogramming of energy production during CD8 T cell responses to viral infections, Ag encounter also promotes cholesterol biosynthesis (Chen et al., 1975;Kidani et al., 2013) and triggers an increase in glutamine uptake and glutaminolysis (Carr et al., 2010;Wang et al., 2011), which fuels FA synthesis through -KetoGlutarate (-KG)dependent citrate production (Gupta et al., 2020;Pearce et al., 2013). T cell activation also redirects glyceraldehyde-3P (G3P) downstream glucose degradation towards the production of p. 3 5-phosphoribose-2P (PRPP) that fuels the pentose-phosphate pathway (PPP) to meet nucleic acid and aromatic amino acid biosynthesis demands (Wang et al., 2011). ...
... Finally, we detected the increased cholesterol production described in activated CD8 T cells (Chen et al., 1975;Kidani et al., 2013) only after 4 dpi, suggesting its role may not be major for the immune synapse formation (Yang et al., 2016) but important for the regulation of the transcriptional activity of Liver X receptor (Bensinger et al., 2008) and Sterol Regulatory Element-Binding Protein (Kidani et al., 2013), in activated T cells (Møller et al., 2022) SM4, 5, 7, 8, 14, 15, 18 or 19, defined in Alghamdi et al. (2021) as 'glycan, glycosaminoglycan, glutamate, glycogen, sialic and fatty acids, beta-alanine and aspartate metabolism or synthesis'. We cannot conclude whether these sections of CD8 T cell metabolism are less affected by cellular differentiation or whether the algorithm fails to evidence them. ...
CD8 T cell proper differentiation during antiviral responses relies on metabolic adaptations. Herein, we investigated global metabolic activity in single CD8 T cells along an in vivo response by estimating metabolic fluxes from single-cell RNA-sequencing data. The approach was validated by the observation of metabolic variations known from experimental studies on global cell populations, while adding temporally detailed information and unravelling yet undescribed sections of CD8 T cell metabolism that are affected by cellular differentiation. Furthermore, inter-cellular variability in gene expression level, highlighted by single cell data, and heterogeneity of metabolic activity 4 days post-infection, revealed a new transition stage accompanied by a metabolic switch in activated cells differentiating into full-blown effectors.
... highlighted that hepatic effector memory CD4 + T cells are depleted, while central memory cells are increased, in proportion to the extent of viremia, displaying higher levels of CCR5 and PD-1. Transcriptomic analyses revealed activated CD4 + T cells (human leukocyte antigen [HLA]-DR transcripts) associated with higher levels of mitoribosome and cholesterol synthesis transcripts, which have long been recognized to be necessary for cell growth and proliferation (74)(75)(76). We also found a strong enrichment of genes that dynamically regulate ER, mitochondria, and lysosomal machinery (reticulophagy and mitophagy). ...
... However, in the absence of extrinsic signals, cell viability is compromised by insufficient nutrient utilization (79). Cholesterol has long been recognized to be an important component of cell membranes and an absolute requirement in cell cycle progression, cell growth, and proliferation (74)(75)(76). Therefore, the requirement for adequate cholesterol is obvious in activated CD4 + T cells (80,81). ...
While liver inflammation is associated with AIDS, little is known so far about hepatic CD4 ⁺ T cells. By using the simian immunodeficiency virus (SIV)-infected rhesus macaque (RM) model, we aimed to characterize CD4 ⁺ T cells. The phenotype of CD4 ⁺ T cells was assessed by flow cytometry from uninfected ( n = 3) and infected RMs, with either SIVmac251 ( n = 6) or SHIVSF162p3 ( n = 6). After cell sorting of hepatic CD4 ⁺ T cells, viral DNA quantification and RNA sequencing were performed.Thus, we demonstrated that liver CD4 ⁺ T cells strongly expressed the SIV coreceptor, CCR5. We showed that viremia was negatively correlated with the percentage of hepatic effector memory CD4 ⁺ T cells. Consistent with viral sensing, inflammatory and interferon gene transcripts were increased. We also highlighted the presence of harmful CD4 ⁺ T cells expressing GZMA and members of TGFB that could contribute to fuel inflammation and fibrosis. Whereas RNA sequencing demonstrated activated CD4 ⁺ T cells displaying higher levels of mitoribosome and membrane lipid synthesis transcripts, few genes were related to glycolysis and oxidative phosphorylation, which are essential to sustain activated T cells. Furthermore, we observed lower levels of mitochondrial DNA and higher levels of genes associated with damaged organelles (reticulophagy and mitophagy). Altogether, our data revealed that activated hepatic CD4 ⁺ T cells are reprogrammed to lipid metabolism. Thus, strategies aiming to reprogram T cell metabolism with effector function could be of interest for controlling viral infection and preventing liver disorders.
IMPORTANCE
Human immunodeficiency virus (HIV) infection may cause liver diseases, associated with inflammation and tissue injury, contributing to comorbidity in people living with HIV. Paradoxically, the contribution of hepatic CD4 ⁺ T cells remains largely underestimated. Herein, we used the model of simian immunodeficiency virus (SIV)-infected rhesus macaques to access liver tissue. Our work demonstrates that hepatic CD4 ⁺ T cells express CCR5, the main viral coreceptor, and are infected. Viral infection is associated with the presence of inflamed and activated hepatic CD4 ⁺ T cells expressing cytotoxic molecules. Furthermore, hepatic CD4 ⁺ T cells are reprogrammed toward lipid metabolism after SIV infection. Altogether, our findings shed new light on hepatic CD4 ⁺ T cell profile that could contribute to liver injury following viral infection.
... For example, in cholesterol replete conditions, T cells will necessarily decrease de novo synthesis to avoid the deleterious consequences of excess cholesterol accumulation. 13 However, T cells must also preserve metabolite flux into the isoprenoid branches to meet cellular demands of proliferation, differentiation, and effector functions. In this section, we discuss signaling networks that engage the mevalonate pathway in activating T cells and metabolite feedback mechanisms that fine tune pathway flux. ...
... The relevancy of these early findings has been confirmed in lymphocyte cell cycle progression. Early lymphocyte studies determined that induction of T cell mitogenesis triggers HMGCR activity and sterol synthesis prior to the onset of DNA synthesis, cell growth and cell division.13,[139][140][141][142][143][144] Inhibition of mevalonate flux abrogates cell cycle progression, evenin cholesterol-rich culture media. ...
The mevalonate pathway is an essential metabolic pathway in T cells regulating development, proliferation, survival, differentiation, and effector functions. The mevalonate pathway is a complex, branched pathway composed of many enzymes that ultimately generate cholesterol and nonsterol isoprenoids. T cells must tightly control metabolic flux through the branches of the mevalonate pathway to ensure sufficient isoprenoids and cholesterol are available to meet cellular demands. Unbalanced metabolite flux through the sterol or the nonsterol isoprenoid branch is metabolically inefficient and can have deleterious consequences for T cell fate and function. Accordingly, there is tight regulatory control over metabolic flux through the branches of this essential lipid synthetic pathway. In this review we provide an overview of how the branches of the mevalonate pathway are regulated in T cells and discuss our current understanding of the relationship between mevalonate metabolism, cholesterol homeostasis and T cell function.
... Subsequent research leaves little doubt that inhibition of HMGR activity, and the resulting lack of cholesterol synthesis, suppresses cell division. Especially provocative are two findings (Chen et al., 1975;Brown and Goldstein, 1980;Heiniger, 1981;Doyle and Kandutsch, 1988). ...
... Carbon flux through the cholesterogenesis pathway, long known as requisite for DNA synthesis and cell proliferation (Chen et al., 1975;Quesney-Huneeus et al., 1983;Fairbanks et al., 1984;Siperstein, 1984) is also recognized to be temporally coordinated with discrete intervals of the four sequential cell cycle phases that occur between successive mitoses (G 0 , G 1 , S, and G 2 ) (Sánchez-Martín et al., 2007). With synchronized cells in culture, carbon flux into de novo cholesterol is low during early G 1 , increases rapidly to peak midway into G 1 , and declines at the G 1 /S interface. ...
Interpreting connections between the multiple networks of cell metabolism is indispensable for understanding how cells maintain homeostasis or transform into the decontrolled proliferation phenotype of cancer. Situated at a critical metabolic intersection, citrate, derived via glycolysis, serves as either a combustible fuel for aerobic mitochondrial bioenergetics or as a continuously replenished cytosolic carbon source for lipid biosynthesis, an essentially anaerobic process. Therein lies the paradox: under what conditions do cells control the metabolic route by which they process citrate? The Warburg effect exposes essentially the same dilemma—why do cancer cells, despite an abundance of oxygen needed for energy-generating mitochondrial respiration with citrate as fuel, avoid catabolizing mitochondrial citrate and instead rely upon accelerated glycolysis to support their energy requirements? This review details the genesis and consequences of the metabolic paradigm of a “truncated” Krebs/TCA cycle. Abundant data are presented for substrate utilization and membrane cholesterol enrichment in tumors that are consistent with criteria of the Warburg effect. From healthy cellular homeostasis to the uncontrolled proliferation of tumors, metabolic alterations center upon the loss of regulation of the cholesterol biosynthetic pathway. Deregulated tumor cholesterogenesis at the HMGR locus, generating enhanced carbon flux through the cholesterol synthesis pathway, is an absolute prerequisite for DNA synthesis and cell division. Therefore, expedited citrate efflux from cholesterol-enriched tumor mitochondria via the CTP/SLC25A1 citrate transporter is fundamental for sustaining the constant demand for cytosolic citrate that fuels the elevated flow of carbons from acetyl-CoA through the deregulated pathway of cholesterol biosynthesis.
... Added to its toxicity when cholesterol is in excess, its cellular concentrations are tightly controlled [28,29]. Tumor cells demand high amounts of cholesterol for proliferation, which can be satisfied by increasing both cholesterol biosynthesis and LDL uptake through the LDL receptor (LDLR) [30][31][32]. Internalized LDL is degraded in late endosomes/lysosomes (LE/L) to release free cholesterol, which leaves these organelles. Moreover, proliferating cells reduce the removal [33] and increase the storage of cholesterol [34]. ...
... Proliferating cells express both a high rate of cholesterol synthesis, with increased HMG-CoA reductase (HMGCR) activity, and elevated LDLR activity [30][31][32]. Indeed, cancer cells harboring mutant p53 have increased transcription of SREBP-modulated genes [92][93][94][95]. ...
Selective estrogen receptor modulators (SERMs) are a class of compounds that bind to estrogen receptors (ERs) and possess estrogen agonist or antagonist actions in different tissues. As such, they are widely used drugs. For instance, tamoxifen, the most prescribed SERM, is used to treat ERα-positive breast cancer. Aside from their therapeutic targets, SERMs have the capacity to broadly affect cellular cholesterol metabolism and handling, mainly through ER-independent mechanisms. Cholesterol metabolism reprogramming is crucial to meet the needs of cancer cells, and different key processes involved in cholesterol homeostasis have been associated with cancer progression. Therefore, the effects of SERMs on cholesterol homeostasis may be relevant to carcinogenesis, either by contributing to the anticancer efficacy of these compounds or, conversely, by promoting resistance to treatment. Understanding these aspects of SERMs actions could help to design more efficacious therapies. Herein we review the effects of SERMs on cellular cholesterol metabolism and handling and discuss their potential in anticancer pharmacology.
... Besides glucose and glutamine influx, T cells also rapidly upregulate a program for the biosynthesis of fatty acids and cholesterol and increased uptake of lipids after TCR stimulation and mitogen stimulation (Chen, et al., 1975, Bensinger, et al., 2008, Kidani, et al., 2013. Cholesterol is a crucial factor as it is required for cellular growth and proliferation as a component of cell membranes (Chen, et al., 1975, Brown and Goldstein, 1974, Chen, et al., 1974. ...
... Besides glucose and glutamine influx, T cells also rapidly upregulate a program for the biosynthesis of fatty acids and cholesterol and increased uptake of lipids after TCR stimulation and mitogen stimulation (Chen, et al., 1975, Bensinger, et al., 2008, Kidani, et al., 2013. Cholesterol is a crucial factor as it is required for cellular growth and proliferation as a component of cell membranes (Chen, et al., 1975, Brown and Goldstein, 1974, Chen, et al., 1974. Moreover, it is important for lipid raft formation by regulating membrane fluidity, which might play a role in T cell activation by regulating immunological synapse stability (He, et al., 2005, Janes, et al., 2000. ...
This review discusses the relevant metabolic pathways and their regulators which show potential for T cell metabolism-based immunotherapy in diseases hallmarked by both metabolic disease and autoimmunity. Multiple therapeutic approaches using existing pharmaceuticals are possible from a rationale in which T cell metabolism forms the hub in dampening the T cell component of autoimmunity in metabolic diseases. Future research into the effects of a metabolically aberrant micro-environment on T cell metabolism and its potential as a therapeutic target for immunomodulation could lead to novel treatment strategies for metabolic disease-associated autoimmunity.
... The fate of immune cells is influenced not only by their cellular cholesterol content [41][42][43], but also by the metabolites produced through this pathway [44]. Th17 cells increase cholesterol uptake and biosynthesis during differentiation, with sterols serving as endogenous RORγ agonists, thereby affecting the Th17 lineage [45]. ...
The skin is a complex and dynamic organ where homeostasis is maintained through the intricate interplay between the immune system and metabolism, particularly cholesterol metabolism. Various factors such as cytokines, inflammatory mediators, cholesterol metabolites, and metabolic enzymes play crucial roles in facilitating these interactions. Dysregulation of this delicate balance contributes to the pathogenic pathways of inflammatory skin conditions, notably psoriasis. In this article, we provide an overview of omics biomarkers associated with psoriasis in relation to cholesterol metabolism. We explore multi-omics approaches that reveal the communication between immunometabolism and psoriatic inflammation. Additionally, we summarize the use of multi-omics strategies to uncover the complexities of multifactorial and heterogeneous inflammatory diseases. Finally, we highlight potential future perspectives related to targeted drug therapies and research areas that can advance precise medicine. This review aims to serve as a valuable resource for those investigating the role of cholesterol metabolism in psoriasis.
... Undoubtedly, the synthesis and efflux of cholesterol is an essential prerequisite for T-lymphocyte activation and proliferation. 63 SREBPs orchestrate lipid synthesis after T-cell activation. 64 Loss of SREBP2 activity impairs the cholesterol homeostasis of CD8 + T cells and contributes to a significant decrease in cell frequency. ...
Aging has a significant impact on the function and metabolism of T cells. Cholesterol, the most important sterol in mammals, is known as the “gold of the body” because it maintains membrane fluidity, rigidity, and signal transduction while also serving as a precursor of oxysterols, bile acids, and steroid hormones. Cholesterol homeostasis is primarily controlled by uptake, biosynthesis, efflux, and regulatory mechanisms. Previous studies have suggested that there are reciprocal interactions between cholesterol metabolism and T lymphocytes. Here, we will summarize the most recent advances in the effects of cholesterol and its derivatives on T‐cell aging. We will furthermore discuss interventions that might be used to help older individuals with immune deficiencies or diminishing immune competence.
... After 3 and 5 days, 100 µL of activated NBT (2 mg/mL NBT:PBS + 1% PMA) was added to each well, and the plate was incubated for 20 min at 37 • C and 5% CO 2 . Next, 70 µL of 1M HCl followed by 50 µL of 2M KOH and 150 µL of DMSO were added, and the absorbance read at 630 nm, with 405 nm as the reference wavelength [27]. The index of differentiation was then evaluated by dividing NBT by MTT. ...
Tyrosol is a phenolic found in extra virgin olive oil (EVOO). In a Maltese monocultivar EVOO, it was present at a concentration of 9.23 ppm. The HL-60 acute myeloid leukaemia cell line, which can be differentiated to both monocytes and neutrophils, was exposed to tyrosol at this concentration and analysed for evidence of differentiation and effects of cytotoxicity. The polyphenol induced a 1.93-fold increase in cellular oxidative activity (p-value 0.044) and enhanced surface expression of CD11b and CD14. This indicates that tyrosol induces monocytic-like differentiation. An RNA-seq analysis confirmed the upregulation of monocyte genes and the loss of neutrophil genes concomitant with the bi-potential promyelocyte precursor moving down the monocytic pathway. A cell cycle analysis showed an accumulation of cells in the Sub G0/G1 phase following tyrosol exposure for 5 days, which coincided with an increase in apoptotic and necrotic markers. This indicates differentiation followed by cell death, unlike the positive monocyte differentiation control PMA. This selective cytotoxic effect following differentiation indicates therapeutic potential against leukaemia.
... These changes are in line with the essential need for cholesterol production for cell-cycle progression, including in lymphocytes. [33][34][35][36][37] To test the proliferative capacity of CISH-deficient ILC2s, we cultured purified ILC2s in vitro for 1 week with IL-2 and IL-7. Under these conditions, KO ILC2s proliferated more (Fig. 4c, d), grew larger and more granular (Fig. 4e), and produced more IL-5 (Fig. 4f, g) and IL-13 (Fig. 4h). ...
Innate lymphoid cells (ILCs) are tissue-resident effectors poised to activate rapidly in response to local signals such as cytokines. To preserve homeostasis, ILCs must employ multiple pathways, including tonic suppressive mechanisms, to regulate their primed state and prevent inappropriate activation and immunopathology. Such mechanisms remain incompletely characterized. Here we show that cytokine-inducible SH2-containing protein (CISH), a suppressor of cytokine signaling (SOCS) family member, is highly and constitutively expressed in type 2 innate lymphoid cells (ILC2s). Mice that lack CISH either globally or conditionally in ILC2s show increased ILC2 expansion and activation, in association with reduced expression of genes inhibiting cell-cycle progression. Augmented proliferation and activation of CISH-deficient ILC2s increases basal and inflammation-induced numbers of intestinal tuft cells and accelerates clearance of the model helminth, Nippostrongylus brasiliensis, but compromises innate control of Salmonella typhimurium. Thus, CISH constrains ILC2 activity both tonically and after perturbation, and contributes to the regulation of immunity in mucosal tissue.
... As proliferation is a key feature of T-cell effector responses, it is expected that their metabolic reprogramming includes a rapid upregulation of the lipid biosynthetic pathways. Accordingly, it has been known for some time that in lymphocytes, both cholesterol import and active CBP are required for cell cycle progression [100,101]. Cholesterol and cholesterol derivatives shape plasma membrane fluidity and lipid raft dynamics, affecting the formation of the immunological synapse and its downstream signalling events, modulating T-cell activation and function [102,103]. But it was not until 2008 that the relationship between sterol metabolism and T-cell proliferation was described. ...
The immune system protects the body against harm by inducing inflammation. During the immune response, cells of the immune system get activated, divided and differentiated in order to eliminate the danger signal. This process relies on the metabolic reprogramming of both catabolic and anabolic pathways not only to produce energy in the form of ATP but also to generate metabolites that exert key functions in controlling the response. Equally important to mounting an appropriate effector response is the process of immune resolution, as uncontrolled inflammation is implicated in the pathogenesis of many human diseases, including allergy, chronic inflammation and cancer. In this review, we aim to introduce the reader to the field of cholesterol immunometabolism and discuss how both metabolites arising from the pathway and cholesterol homeostasis are able to impact innate and adaptive immune cells, staging cholesterol homeostasis at the centre of an adequate immune response. We also review evidence that demonstrates the clear impact that cholesterol metabolism has in both the induction and the resolution of the inflammatory response. Finally, we propose that emerging data in this field not only increase our understanding of immunometabolism but also provide new tools for monitoring and intervening in human diseases, where controlling and/or modifying inflammation is desirable.
... Interestingly, it is also likely that DC activation through TLR is associated with a high level of fatty acid synthesis, and this metabolic pathway has important roles in the antigen presentation process [22]. B and T cell activation through their antigen receptors is also associated with an increased level of fatty acid synthesis, which is necessary to produce inflammatory cytokines and proliferation of the cells [38,39]. ...
Recent advances in cancer immunotherapy have raised hopes for treating cancers that are resistant to conventional therapies. Among the various immunotherapy methods, the immune checkpoint (IC) blockers were more promising and have paved their way to the clinic. Tumor cells induce the expression of ICs on the immune cells and derive them to a hyporesponsive exhausted phenotype. IC blockers could hinder immune exhaustion in the tumor microenvironment and reinvigorate immune cells for an efficient antitumor response. Despite the primary success of IC blockers in the clinic, the growing numbers of refractory cases require an in-depth study of the cellular and molecular mechanisms underlying IC expression and function. Immunometabolism is recently found to be a key factor in the regulation of immune responses. Activated or exhausted immune cells exploit different metabolic pathways. Tumor cells can suppress antitumor responses via immunometabolism alteration. Therefore, it is expected that concurrent targeting of ICs and immunometabolism pathways can cause immune cells to restore their antitumor activity. In this review, we dissected the reciprocal interactions of immune cell metabolism with expression and signaling of ICs in the tumor microenvironment. Recent findings on dual targeting of ICs and metabolic checkpoints have also been discussed.
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... Interestingly, it is also likely that DC activation through TLR is associated with a high level of fatty acid synthesis, and this metabolic pathway has important roles in the antigen presentation process [22]. B and T cell activation through their antigen receptors is also associated with an increased level of fatty acid synthesis, which is necessary to produce inflammatory cytokines and proliferation of the cells [38,39]. ...
Recent advances in cancer immunotherapy have raised hopes for treating cancers that are resistant to conventional therapies. Among the various immunotherapy methods, the immune checkpoint (IC) blockers were more promising and have paved their way to the clinic. Tumor cells induce the expression of ICs on the immune cells and derive them to a hyporesponsive exhausted phenotype. IC blockers could hinder immune exhaustion in the tumor microenvironment and reinvigorate immune cells for an efficient antitumor response. Despite the primary success of IC blockers in the clinic, the growing numbers of refractory cases require an in-depth study of the cellular and molecular mechanisms underlying IC expression and function. Immunometabolism is recently found to be a key factor in the regulation of immune responses. Activated or exhausted immune cells exploit different metabolic pathways. Tumor cells can suppress antitumor responses via immunometabolism alteration. Therefore, it is expected that concurrent targeting of ICs and immunometabolism pathways can cause immune cells to restore their antitumor activity. In this review, we dissected the reciprocal interactions of immune cell metabolism with expression and signaling of ICs in the tumor microenvironment. Recent findings on dual targeting of ICs and metabolic checkpoints have also been discussed.
... Comprehensive analysis revealed that these dysregulated lipid metabolites were mainly involved in glycerophospholipid metabolism and sphingolipid metabolism (Fig. 1D). We also noticed the dysregulation in fatty acid biosynthesis, which was reported to be necessary for the proliferation of activated B cells and T cells 17,18 . ...
Background: Chronic graft-versus-host disease (cGVHD) remains a common threat after allogeneic hematopoietic cell transplantation (allo-HSCT), and ocular manifestations occur in up to 60-90% of cGVHD patients.
Objectives: To reveal major metabolic dysregulation and to determine tear metabolites as potential biomarkers for ocular cGVHD.
Study Design: Twenty-three ocular cGVHD and sixteen control tear samples were collected for this study. Differential metabolites were identified using a liquid chromatography-mass spectrometry system. Spearman's test was used to analyze the correlation between metabolites and ophthalmic indexes [NIH eye score, fluorescein tear film break-up time (T-BUT), corneal fluorescein staining (CFS), and Schirmer's test]. Receiver operating characteristic (ROC) curve was analyzed to evaluate the prediction potential of identified metabolites for ocular cGVHD.
Results: Differential metabolites were mainly observed in lipid metabolites, and we highlighted the lipid dysregulation in glycerophospholipid metabolism, sphingolipid metabolism, and biosynthesis of unsaturated fatty acids. In glycerophospholipid metabolism, phosphatidylcholine (34:1) [PC (34:1)] exhibited the strongest correlation with NIH eye score (r = 0.80), T-BUT (r = 0.79), CFS (r = 0.77), and Schirmer's test (r = 0.69). In sphingolipid metabolism, sphingomyelin (SM) was the most consistent with T-BUT (r = 0.74) and CFS (r = 0.71), while lactosylceramide (LacCer) was the most consistent with NIH eye score (r = 0.76) and Schirmer's test ((r = 0.64). In biosynthesis of unsaturated fatty acids, docosahexaenoic acid (DHA) had the highest correlation with NIH eye score (r = 0.73), T-BUT (r = 0.60), CFS (r = 0.67) and Schirmer's test (r = 0.67) (P < 0.0001 for all). ROC analysis revealed that area under the curve (AUC) values for PC (34:1) (AUC = 0.967), LacCer (AUC = 0.946), SM (AUC = 0.932), and DHA (AUC = 0.929) were significantly correlated with cGVHD (P < 0.0001 for all).
Conclusions: Our study identified PC (34:1), SM, LacCer, and DHA as promising tear biomarkers to indicate metabolic dysregulation and ophthalmic manifestations in ocular cGVHD.
... Fatty acid synthesis was observed to be upregulated during TLR-mediated dendritic cell activation (Everts et al., 2014). In addition, fatty acid synthesis was also found to be necessary for cell proliferation after the activation of T and B cells via antigen receptors (Chen et al., 1975;Dufort et al., 2014). Accompanied by the present results in correlation analysis and pathway analysis, those of acylcarnitines were significantly positively associated with proinflammatory factors in the colon (Figure 8), as well as connected with CoA biosynthesis. ...
Inflammatory bowel disorder is accompanied by the destruction of immunity homeostasis, gut microbiota perturbation, and chronic inflammatory liver diseases. Butyrate is known as a primary energy source for colonocytes and functional substances for mitigating pathological features of colitis. However, it is still unclear whether butyrate alleviates colitis progression by regulation of microbiota and metabolism in the gut–liver axis. In the present study, we aimed to determine the role of microbiota and metabolism of the gut–liver axis in ameliorating lipopolysaccharide (LPS)-induced colitis in piglets using protected butyrate administration. Eighteen crossbred male piglets were weaned at 30 days old and were randomly allocated to three treatments, with CON (basal diet), LPS (basal diet + LPS), and BT-LPS (basal diet + 3.0 g/kg protected butyrate + LPS). On days 19 and 21, piglets in the LPS and BT-LPS groups were intraperitoneally challenged with LPS at 100 μg/kg body weight. Butyrate administration significantly decreased LPS-induced rise in the clinical score of piglets and colonic histological scores and reduced the susceptibility to LPS-induced severe inflammatory response by decreasing proinflammatory (IL-1β, IL-6, IL-8, and TNF-α) cytokines. Butyrate supplementation accelerated the prevalence of Faecalibacterium and Lactobacillus by enhancing the tricarboxylic acid (TCA) cycle of colonocytes. Dietary supplementation with protected butyrate significantly targeted increased concentrations of butyric acid in the colon and portal venous circulation, and enhanced the TCA cycle in the gut–liver axis by mobilizing amino acid and vitamin B group as a coenzyme. Meanwhile, during this progress, LPS increased fatty acid synthesis that was reversed by butyrate treatment, which was reflected by decreased acylcarnitines. Butyrate-reshaped colonic microbial community and metabolism in the gut–liver axis contributed to morphology integrity and immunity homeostasis by promoting anti-inflammatory (IL-10 and TGF-β) cytokines and suppressing inflammatory mediator hypoxia-inducible factor 1α and its downstream response elements cyclooxygenase 2 and inducible nitric oxide synthase. These results identified the pivotal role of colonic microbiota and metabolism in the gut–liver axis for alleviating inflammatory progression and possible therapeutic targets.
... Transdifferentiation of b-cells to a-cells might also contribute to the b-cell mass reduction. Complete inhibition of HMGCR by pharmacological doses of statins inhibits cell replication in many types of cells, including b-cell-like MIN6 cells (9,23). Indeed, we showed that deletion of Hmgcr in the liver leads to fatal hypoglycemia associated with severe liver dysfunction (15) and that deletion of Hmgcr in the skeletal muscles leads to rhabdomyolysis (16). ...
Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), statins, which are used to prevent cardiovascular diseases, are associated with a modest increase in the risk of new-onset diabetes mellitus. To investigate the role of HMGCR in the development of β cells and glucose homeostasis, we deleted Hmgcr in a β cell-specific manner by using the Cre-loxP technique. Mice lacking Hmgcr in β cells (β-KO) exhibited hypoinsulinemic hyperglycemia as early as postnatal day 9 (P9) due to decreases in both β cell mass and insulin secretion. Ki67 positive cells were reduced in β-KO mice at P9, thus β cell mass reduction was caused by proliferation disorder immediately after birth. The mRNA expression of neurogenin3 (Ngn3), which is transiently expressed in endocrine progenitors of the embryonic pancreas, was maintained despite a striking reduction in the expression of β cell-associated genes, such as insulin, Pancreatic and duodenal homeobox 1 (Pdx1) and MAF BZIP transcription factor A (Mafa) in the islets from β-KO mice. Histological analyses revealed dysmorphic islets with markedly reduced numbers of β cells, some of which were also positive for glucagon. In conclusion, HMGCR plays critical roles not only in insulin secretion but also in the development of β cells in mice.
... T cells rapidly increase their cholesterol stores and the expression of cholesterol biosynthesis genes upon activation. 40,[80][81][82] Though a major role for this increase in cholesterol biosynthesis is to support membrane production for proliferation, the pathway is also appreciated to control T cell signaling at multiple levels. ...
The maintenance of organismal homeostasis requires partitioning and transport of biochemical molecules between organ systems, their composite cells, and subcellular organelles. Although transcriptional programming undeniably defines the functional state of cells and tissues, underlying biochemical networks are intricately intertwined with transcriptional, translational, and post-translational regulation. Studies of the metabolic regulation of immunity have elegantly illustrated this phenomenon. The cells of the immune system interface with a diverse set of environmental conditions. Circulating immune cells perfuse peripheral organs in the blood and lymph, patrolling for pathogen invasion. Resident immune cells remain in tissues and play more newly appreciated roles in tissue homeostasis and immunity. Each of these cell populations interacts with unique and dynamic tissue environments, which vary greatly in biochemical composition. Furthermore, the effector response of immune cells to a diverse set of activating cues requires unique cellular adaptations to supply the requisite biochemical landscape. In this review, we examine the role of spatial partitioning of metabolic processes in immune function. We focus on studies of lymphocyte metabolism, with reference to the greater immunometabolism literature when appropriate to illustrate this concept.
... It was found that increased fatty acid synthesis (FAS) is necessary for DC activation and their subsequent stimulation of CD8+ T cells [207]. FAS is also important for both T cells and B cells, especially for cell proliferation after activation [208,209]. Intriguingly, T cell-specific deletion of acetyl-CoA carboxylase 1 (ACC1), the rate-limiting enzyme in FAS, was found to reduce blasting efficacy along with a lower accumulation of antigen-specific CD8+ T cells, which could be reversed by supplementing them with exogenous fatty acids [210]. However, neither pharmacological nor genetic inhibition of ACC1 in CD4+ T cell subsets seems to affect Treg cell generation and function [211]. ...
In the relatively short history of anti-tumor treatment, numerous medications have been developed against a variety of targets. Intriguingly, although many anti-tumor strategies have failed in their clinical trials, metformin, an anti-diabetic medication, demonstrated anti-tumor effects in observational studies and even showed its synergistic potential with immune checkpoint inhibitors (ICIs) in subsequent clinical studies. Looking back from bedside-to-bench, it may not be surprising that the anti-tumor effect of metformin derives largely from its ability to rewire aberrant metabolic pathways within the tumor microenvironment. As one of the most promising breakthroughs in oncology, ICIs were also found to exert their immune-stimulatory effects at least partly via rewiring metabolic pathways. These findings underscore the importance of correcting metabolic pathways to achieve sufficient anti-tumor immunity. Herein, we start by introducing the tumor microenvironment, and then we review the implications of metabolic syndrome and treatments for targeting metabolic pathways in anti-tumor therapies. We further summarize the close associations of certain aberrant metabolic pathways with impaired anti-tumor immunity and introduce the therapeutic effects of targeting these routes. Lastly, we go through the metabolic effects of ICIs and conclude an overall direction to manipulate metabolic pathways in favor of anti-tumor responses.
... PHA crosslinks glycosylated surface receptors such as the T cell receptor (TCR), leading to upregulation of glycolysis within 3 hours, particularly in the T cell population. [58][59][60][61] Murine T cells stimulated via the TCR upregulate surface GLUT1 receptors within 2-4 hr, an early hallmark of T cell activation. 62 Additionally, increased hexokinase activity has been observed after 2 hr PHA stimulation of mixed lymphocytes. ...
Highly proliferative cells depend heavily on glycolysis as a source of energy and biological precursor molecules, and glucose uptake is a useful readout of this aspect of metabolic activity. Glucose uptake is commonly quantified by using flow cytometry for cell cultures and positron emission tomography for organs in vivo. However, methods to detect spatiotemporally resolved glucose uptake in intact tissues are far more limited, particularly those that can quantify changes in uptake over time in specific tissue regions and cell types. Using lymph node metabolism as a case study, we developed a novel assay of dynamic and spatially resolved glucose uptake in living tissue by combining ex vivo tissue slice culture with a fluorescent glucose analogue. Live slices of murine lymph node were treated with the glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino]-2-deoxyglucose (2-NBDG). Incubation and washing parameters were optimized to differentiate glucose uptake in activated versus naive lymphocytes. Confocal microscopy of treated tissues confirmed that the 2-NBDG signal was intracellular. The assay was readily multiplexed with live immunofluorescence labelling to generate maps of 2-NBDG uptake across tissue regions, revealing highest uptake in T cell-dense regions. Uptake was predominantly localized to lymphocytes rather than stromal cells. Because 2-NBDG washed out of the tissue over time, the assay was repeatable in the same slices before and after T cell activation to reveal which tissue regions were most responsive to stimulation. We anticipate that this assay will serve as a broadly applicable, user-friendly platform to quantify dynamic metabolic activities in complex tissue microenvironments.
... LDL receptor expression by mitogenic surge (in presence of insulin) has been found to be under control of sterol regulated feedback mechanisms in cells (Gopalakrishnan and Chandra , 2006). Chen et al., (1975) first showed that phytohemagglutinin-stimulated mouse lymphocytes underwent a six fold increase in acetate incorporation into cholesterol 24 h before the cells entered the S-phase of the cell cycle. Unlike biological mitogens the SREBP mediated feedback regulation remains declined in presence of carcinogens. ...
Background
B cells are specific antibody generating cells which respond to foreign intruders in the circulation. The purpose of this study was to compare the relative immunogenic potentials of three well established agent types viz. an immunogen, a mitogen and a carcinogen, by following B cell responses to their presence in a mouse model system.
Methods
Mice were treated with tetanus toxoid (immunogen), poke weed mitogen (typical mitogen), and benzo-α-pyrene (carcinogen) and generated B cell populations were determined in isolated splenic lymphocytes (splenocytes) by flow cytometry using specific anti-B cell marker antibodies. Flow cytometric estimation of LDL receptor (LDLR) expression, along with associated B cell markers, was also conducted. Kit based estimation of serum IgG, western blotting for LDLR estimation on total splenocytes and spectrometry for cholesterol and serum protein estimation were further undertaken. Student’s T-tests and one way ANOVA followed by the Bonferroni method were employed for statistical analysis.
Results
The mitogen was found to better stimulate B cell marker expression than the immunogen, although the latter was more effective at inducing antibody production. The chemical carcinogen benzo-α-pyrene at low concentration acted potentially like a mitogen but almost zero immunity was apparent at a carcinogenic dose, with a low profile for LDLR expression and intracellular cholesterol.
Conclusion
The findings in our study demonstrate an impact of concentration of BaP on generation of humoral immunity. Probably by immunosuppression through restriction of B-cell populations and associated antibodies, benzo-α-pyrene may exerts carcinogenicity. The level of cholesterol was found to be a pivotal target.
... 26,48 Primarily, isotopomer-enrichment studies showed that activation of lymphocytes results in a rapid increase in new biosynthesis of cholesterol and fatty acids. 49 In addition, pharmacological inhibition of the enzyme HMGCR by statins inhibited anti-CD3-induced T-cell mitogenesis in a dose-dependent manner. 24 Finally, Hedrick and colleagues demonstrated that robust cholesterol accumulation by genetic depletion of the cholesterol transporter, ABCG1, not only did not cause ER stressinduced apoptosis, it actually provided a proliferative advantage for naive T cells. ...
Acquisition of a "quiescence program" by naïve T cells is important to provide a stress-free environment and resistance to apoptosis while preserving their responsiveness to activating stimuli. Therefore, the survival and proper function of naïve T cells depends on their ability to maintain quiescence. Recently we demonstrated that by preventing chronic unresolved endoplasmic reticulum (ER) stress, Schlafen2 (Slfn2) maintains a stress-free environment to conserve a pool of naïve T cells ready to respond to a microbial invasion. These findings strongly suggest an intimate association between quiescence and stress signaling. However, the connection between ER stress conditions and loss of T cell quiescence is unknown. Here we demonstrate that homeostasis of cholesterol and lipids, is disrupted in T cells and monocytes from elektra mice with higher levels of lipid rafts and lipid droplets found in these cells. Moreover, elektra T cells had elevated levels of free cholesterol and cholesteryl ester due to increased de novo synthesis and higher levels of the enzyme HMGCR. As cholesterol plays an important role in the transition of T cells from resting to active state, and ER regulates cholesterol and lipid synthesis, we suggest that regulation of cholesterol levels through the prevention of ER stress is an essential component of the mechanism by which Slfn2 regulates quiescence.
... SCD and FADS1, which encode fatty acid desaturases, were both up-regulated in response to inflammatory challenge, despite small variations. The up-regulation of these lipid-biosynthetic pathways was in accordance with early isotopomer-enrichment studies that demonstrated that the activation of lymphocytes results in a rapid increase in the renewed biosynthesis of cholesterol and fatty acids 20 . According to this study, the synthesis of cholesterol could be an essential prerequisite for successful initiation and completion of the cell cycle in lymphocytes. ...
Negative Energy Balance (NEB) is considered to increase susceptibility to mastitis. The objective of this study was to improve our understanding of the underlying mechanisms by comparing transcriptomic profiles following NEB and a concomitant mammary inflammation. Accordingly, we performed RNA-seq analysis of blood cells in energy-restricted ewes and control-diet ewes at four different time points before and after intra mammary challenge with phlogogenic ligands. Blood leucocytes responded to NEB by shutting down lipid-generating processes, including cholesterol and fatty acid synthesis, probably under transcriptional control of SREBF 1. Furthermore, fatty acid oxidation was activated and glucose oxidation and transport inhibited in response to energy restriction. Among the differentially expressed genes (DEGs) in response to energy restriction, 64 genes were also differential in response to the inflammatory challenge. Opposite response included the activation of cholesterol and fatty acid synthesis during the inflammatory challenge. Moreover, activation of glucose oxidation and transport coupled with the increase of plasma glucose concentration in response to the inflammatory stimuli suggested a preferential utilization of glucose as the energy source during this stress. Leucocyte metabolism therefore undergoes strong metabolic changes during an inflammatory challenge, which could be in competition with those induced by energy restriction.
... Fatty acid synthesis was found to be upregulated during Toll-like receptor (TLR)-mediated DC activation, and this increased fatty acid synthesis was necessary for DC activation and their stimulation of CD8 + T cell responses 20 . Fatty acid synthesis is also key to the cell intrinsic function of T cells and B cells; synthesis of fatty acids and sterols has been shown to be necessary for cell proliferation after the activation of these cells through their antigen receptors 55,56 . Recent work demonstrated that T cellspecific deletion of acetyl-CoA carboxylase 1 (ACC1) -the rate-limiting enzyme in fatty acid synthesis -results in reduced blasting efficacy and lower accumulation of antigenspecific CD8 + T cells. ...
In recent years a substantial number of findings have been made in the area of immunometabolism, by which we mean the changes in intracellular metabolic pathways in immune cells that alter their function. Here, we provide a brief refresher course on six of the major metabolic pathways involved (specifically, glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, fatty acid oxidation, fatty acid synthesis and amino acid metabolism), giving specific examples of how precise changes in the metabolites of these pathways shape the immune cell response. What is emerging is a complex interplay between metabolic reprogramming and immunity, which is providing an extra dimension to our understanding of the immune system in health and disease.
Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.
Intermediary metabolism of tumors is characterized, in part, by a dysregulation of the cholesterol biosynthesis pathway at its rate-controlling enzyme providing the molecular basis for tumor membranes (mitochondria, plasma membrane) to become enriched with cholesterol (Bloch, 1965; Feo et al., 1975; Brown and Goldstein, 1980; Goldstein and Brown, 1990). Cholesterol enriched tumor mitochondria manifest preferential citrate export, thereby providing a continuous supply of substrate precursor for the tumor’s dysregulated cholesterogenesis via a “truncated” Krebs/TCA cycle (Kaplan et al., 1986; Coleman et al., 1997). Proliferating tumors shed elevated amounts of plasma membrane-derived extracellular vesicles (pmEV) compared with normal tissues (van Blitterswijk et al., 1979; Black, 1980). Coordination of these metabolic phenomena in tumors supports the enhanced intercalation of cholesterol within the plasma membrane lipid bilayer’s cytoplasmic face, the promotion of outward protrusions from the plasma membrane, and the evolution of cholesterol enriched pmEV. The pmEV shed by tumors possess elevated cholesterol and concentrated cell surface antigen clusters found on the tumor cells themselves (Kim et al. (2002)). Upon exfoliation, saturation of the extracellular milieu with tumor-derived pmEV could allow early onset mammalian immune surveillance mechanisms to become “blind” to an evolving cancer and lose their ability to detect and initiate strategies to destroy the cancer. However, a molecular mechanism is lacking that would help explain how cholesterol enrichment of the pmEV inner lipid bilayer might allow the tumor cell to evade the host immune system. We offer a hypothesis, endorsed by published mathematical modeling of biomembrane structure as well as by decades of in vivo data with diverse cancers, that a cholesterol enriched inner bilayer leaflet, coupled with a logarithmic expansion in surface area of shed tumor pmEV load relative to its derivative cancer cell, conspire to force exposure of otherwise unfamiliar membrane integral protein domains as antigenic epitopes to the host’s circulating immune surveillance system, allowing the tumor cells to evade destruction. We provide elementary numerical estimations comparing the amount of pmEV shed from tumor versus normal cells.
Productive T cell responses to infection and cancer rely on coordinated metabolic reprogramming and epigenetic remodeling among the immune cells. In particular, T cell effector and memory differentiation, exhaustion, and senescence/aging are tightly regulated by the metabolism-epigenetics axis. In this review, we summarize recent advances of how metabolic circuits combined with epigenetic changes dictate T cell fate decisions and shape their functional states. We also discuss how the metabolic-epigenetic axis orchestrates T cell exhaustion and explore how physiological factors, such as diet, gut microbiota, and the circadian clock, are integrated in shaping T cell epigenetic modifications and functionality. Furthermore, we summarize key features of the senescent/aged T cells and discuss how to ameliorate vaccination- and COVID-induced T cell dysfunctions by metabolic modulations. An in-depth understanding of the unexplored links between cellular metabolism and epigenetic modifications in various physiological or pathological contexts has the potential to uncover novel therapeutic strategies for fine-tuning T cell immunity.
Les lymphocytes T CD4+ Foxp3+ régulateurs (Treg) participent à la régulation de l’activité du système immunitaire et sont essentiel au maintien de la tolérance immune. Ces cellules sont ainsi bénéfiques lors de pathologies auto-immunes tandis que leur action anti-inflammatoire favorise la croissance tumorale. Comprendre le fonctionnement des Treg constitue donc un axe majeur pour le développement de nouvelles stratégies thérapeutiques applicables à ces deux types de pathologies. Au cours de ce travail, nous nous sommes intéressés au métabolisme des Treg étant donné que les informations disponibles à ce jour font encore l’objet de controverse. A l’inverse des lymphocytes T conventionnels (Tconv), qui utilisent un métabolisme de type anabolique sous le control de mTOR, les Treg sont aujourd’hui considéré comme des cellules présentant un métabolisme de type catabolique favorisé par l’AMPK. Afin de vérifier cette hypothèse, nous avons utilisés des modèles murins de délétion conditionnelle permettant la délétion génique d’AMPK ou mTOR, spécifiquement dans les Treg. L’étude de ces souris a permis de mettre en évidence un rôle jusqu’ici insoupçonné de mTOR dans la stabilité et la migration des Treg ainsi qu’une implication de l’AMPK dans la capacité des Treg à inhiber la réponse anti-tumorale. Ces travaux remettent ainsi en question les considérations actuelles sur le métabolisme des Treg et ouvrent la voie vers une meilleure compréhension du métabolisme de ces cellules à l’homéostasie et en condition pathologique.
Checkpoint-based immunotherapies, such as programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) inhibitors, have shown promising clinical outcomes in many types of cancers. Unfortunately, the response rate of immune checkpoint inhibitors is low. It is very important to discover novel therapeutic targets and prognostic biomarkers. Cholesterol metabolism has been demonstrated to be related to the occurrence and development of a variety of tumors and may provide a new breakthrough in the development of immunotherapy. First of all, cholesterol metabolism in the tumor microenvironment affects the function of tumor-infiltrating immune cells. In addition, intracellular cholesterol homeostasis is an important regulator of immune cell function. Furthermore, drugs that act on cholesterol metabolism affect the efficacy of immunotherapy. What is more, peripheral blood cholesterol level can be a biomarker to predict the efficacy of immunotherapy. In this review, we aimed to explore the potential role of cholesterol metabolism on immunotherapy. By summarizing the major findings of recent preclinical and clinical studies on cholesterol metabolism in immunotherapy, we suggested that cholesterol metabolism could be a potential therapeutic target and a prognostic biomarker for immunotherapy.
The mevalonate pathway is responsible for the synthesis of isoprenoids, including sterols and other metabolites that are essential for diverse biological functions. Cholesterol, the main sterol in mammals, and non-sterol isoprenoids are in high demand by rapidly dividing cells. As evidence of its importance, many cell signaling pathways converge on the mevalonate pathway and these include those involved in proliferation, tumor-promotion, and tumor-suppression. As well as being a fundamental building block of cell membranes, cholesterol plays a key role in maintaining their lipid organization and biophysical properties, and it is crucial for the function of proteins located in the plasma membrane. Importantly, cholesterol and other mevalonate derivatives are essential for cell cycle progression, and their deficiency blocks different steps in the cycle. Furthermore, the accumulation of non-isoprenoid mevalonate derivatives can cause DNA replication stress. Identification of the mechanisms underlying the effects of cholesterol and other mevalonate derivatives on cell cycle progression may be useful in the search for new inhibitors, or the repurposing of preexisting cholesterol biosynthesis inhibitors to target cancer cell division. In this review, we discuss the dependence of cell division on an active mevalonate pathway and the role of different mevalonate derivatives in cell cycle progression.
Background
Allergen-specific immunotherapy (AIT) is a disease-modifying treatment that induces long-term T cell tolerance.
Objective
To evaluate the role of circulating CXCR5⁺PD-1⁺T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous (SCIT) and sublingual (SLIT) immunotherapy and the accompanying changes in their chromatin landscape.
Methods
Phenotype and function of cTFH cells were initially evaluated in grass pollen-allergics (GPA, n= 28) and non-atopic controls (NAC, n=13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n=12), GPA (n=14), SCIT (n=10) and SLIT (n=8)-treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by ATAC-seq to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT and SLIT.
Results: cTFH cells were shown to be distinct from TH2 and TH2A cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH cell proliferation in GPA but not in NAC (P<.05). cTFH cells were higher in GPA compared to NAC and were lower in SCIT and SLIT (P<.01). Time-dependent induction of IL-4, IL-21 and IL-6 were observed in nasal mucosa following intranasal allergen challenge in GPA but not in SCIT and SLIT groups. TFR and IL-10⁺ cTFH cells were induced in SCIT and SLIT (all, P<.01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups.
Conclusion
For the first time, we showed dysregulation of cTFH cells in GPA compared to NAC, SCIT and SLIT and induction of TFR and IL-10⁺ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following AIT in cTFH and TFR cells.
Highly proliferative cells depend heavily on glycolysis as a source of energy and biological precursor molecules, and glucose uptake is a useful readout of this aspect of metabolic activity. Glucose uptake is commonly quantified by using flow cytometry for cell cultures and positron emission tomography for organs in vivo. However, methods to detect spatiotemporally resolved glucose uptake in intact tissues are far more limited, particularly those that can quantify changes in uptake over time in specific tissue regions and cell types. Using lymph node metabolism as a case study, we developed an optimized method to detect dynamic and spatially resolved glucose uptake in living tissue by combining ex vivo tissue slice culture with a fluorescent glucose analogue. Live slices of murine lymph node were treated with the glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino]-2-deoxyglucose (2-NBDG). Incubation parameters were optimized to differentiate glucose uptake in activated versus naïve lymphocytes. Regional glucose uptake could be imaged at both the tissue level, by widefield microscopy, and at the cellular level, by confocal microscopy. Furthermore, the glucose assay was readily multiplexed with live immunofluorescence labelling to generate maps of 2-NBDG uptake across tissue regions, revealing highest uptake in T cell-dense regions. The signal was predominantly intracellular and localized to lymphocytes rather than stromal cells. Finally, we demonstrated that the assay was repeatable in the same slices, and imaged the dynamic distribution of glucose uptake in response to ex vivo T cell stimulation for the first time. We anticipate that this method will serve as a broadly applicable, user-friendly platform to quantify dynamic metabolic activities in complex tissue microenvironments.
The cell cycle is a sequential multi-step process essential for growth and proliferation of cells comprising multicellular organisms. A number of nuclear and cytoplasmic proteins are known to modulate the cell cycle. Yet, the role of lipids, membrane organization and physical properties in cell cycle progression remains largely elusive. Membrane dipole potential is an important physicochemical property and originates due to the electrostatic potential difference within the membrane because of nonrandom arrangement of amphiphile dipoles and water molecules at the membrane interface. In this work, we explored the modulation of membrane dipole potential in various stages of the cell cycle in CHO-K1 cells. Our results show that membrane dipole potential is highest in G1 phase relative to S and G2/M phases. This was accompanied by regulation of membrane cholesterol content in the cell cycle. The highest cholesterol content was found in G1 phase with considerable reduction in cholesterol in S and G2/M phases. Interestingly, we noted a similarity in the dependence of membrane dipole potential and cholesterol with progress of cell cycle. In addition, we observed an increase in neutral lipid (which contain esterified cholesterol) content as cells progressed from G1 to G2/M via S phase of the cell cycle. Importantly, we further observed a cell cycle dependent reduction in ligand binding activity of serotonin1A receptors expressed in CHO-K1 cells. To the best of our knowledge, these results constitute the first report of cell cycle dependent modulation of membrane dipole potential and activity of a neurotransmitter receptor belonging to the G protein-coupled receptor family. We envision that understanding the basis of cell cycle events from a biophysical perspective would result in a deeper appreciation of cell cycle and its regulation in relation to cellular function.
Like all dividing cells, naïve T cells undergo a predictable sequence of events to enter the cell cycle starting from G0 and progressing to G1, S and finally G2/M. This methodical series of steps ensures the fidelity in generating two identical T cells during a single round of division. To achieve this, T cells must activate or inactivate metabolic pathways at discrete times during each phase of the cell cycle. This permits the generation of substrates to support biosynthesis, bioenergetics, and the epigenetic changes required for proper differentiation and function. The precursors that feed into these pathways are often shared, highlighting the complex relationship between metabolism and cellular processes that are essential to lymphocytes. It is therefore not surprising that different T cell subtypes exhibit unique metabolic dependencies that change as they mature and undergo specialized differentiation programs. The importance of how metabolism influences T cells is underscored by the emerging field of cancer immunotherapy, where autologous T cells can be manufactured ex vivo then infused as a form of curative treatment for human cancers. This review will highlight some of the recent knowledge on T lymphocyte metabolism and give a perspective on the practical implications for cellular‐based immunotherapy. This article is protected by copyright. All rights reserved.
Recent epidemiological and preclinical studies have shown that the steroidal lipid cholesterol is a clinically relevant therapeutic target in prostate cancer. This review summarizes the findings from human studies, as well as from animal models and cell biology approaches, which suggest that high circulating cholesterol can increase the risk of aggressive prostate cancer, while pharmacological cholesterol lowering may be protective against incident or advanced disease. A variety of molecular processes are described that have been implicated experimentally in this protective scenario or are otherwise plausibly connected. There is now sufficient experimental and observational evidence in humans to prospectively apply cholesterol- targeting strategies in selected patients to inhibit prostate cancer progression to the metastatic form of the disease.
CD4+ T helper 17 cells (Th17) acquire specific effector functions in response to activation and instructional signals. Accumulating evidence indicates that specific cellular lipid metabolic pathways play essential roles in regulating the differentiation and function of Th17 cells. Mechanistic studies reveal that metabolic fluxes through both the cholesterol and long chain fatty acid biosynthetic pathways are important in controlling RORγ transcriptional activity through their ability to generate lipid ligands of RORγ. Genetic and pharmacologic studies demonstrate that altering the flux through these lipid biosynthetic pathways impacts the generation of IL-17 as well as the balance of Th17 and CD4+ regulatory T cells (Tregs). In this mini-review, we briefly introduce the mechanics of cholesterol and long chain fatty acid biosynthesis. We also discuss the evidence underlying the unique role that these lipid metabolic pathways play in intrinsically regulating the fate and function of Th17 cells under normal and pathogenic conditions.
1) The effect of 4 weeks administration with immunomodulator PSK (Krestin) on the lipoprotein lipids contents in serum, was investigated in 10 patients with hyperlipidemia including three FH heterozygous patients. There was a significant decrease of T-cholesterol, β-lipoprotein in serum. HDL-cho, and triglyceride showed no significant alterations.
2) In a series of in vitro experiments, the effects of PSK on the LDL receptor activity of normal and FH heterozygous fibroblasts were investigated. In normal subject, PSK increased the uptake of ¹²⁵I-LDL by+31% at 100μg/ml, while in FH heterozygous cells, the uptake was increased by +117% at 10μg/ml.
3) One of the reasons why β-lipoprotein in serum is decreased in patients of hyperlipidemia by PSK administration is considered to be that PSK enhanced the LDL receptor activity of fibroblasts. It was also suggested that PSK has stimulated more effectively LDL receptor activity of fibroblasts from hyperlipidemia patients than from normal subject.
For the past several decades, the quality and quantity of dietary lipid has come under close scrutiny regarding its role in the pathogenesis of coronary heart disease and cancer at any site, the two leading causes of moribidity and mortality in affluent societies. This concern over dietary lipids has resulted in a serious controversy among health professionals who are in disagreement as to whether a reduction in dietary lipid from some stated average of 40% to 30%, or lower, of total calories is beneficial, harmful or warranted. Even a cursory review of the current literature will provide support for each of these positions.
The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique features that make it of special interest in relationship to cell replication. Principal products of the biosynthetic pathway, cholesterol, dolichyl phosphate and coenzyme Q, are essential, respectively, for plasma membrane stability and function, for arginine-linked glycoprotein synthesis and for hydrogen transport. Prenylation of important proteins such as lamin B1 and the ras oncogene protein2 may be required for their actions during cell replication, thus, together the products of this pathway influence cell structure and many cellular functions. Except for the conversion of cholesterol to other steroids in liver and endocrine cells, the polyprenyl products of the pathway do not seem to be degraded and, in replicating cells, their synthesis appears to be correlated with cell division3–5. The additional cholesterol needed for cell replication can be taken up from the extracellular medium if it is available6. But the requirement for dolichyl phosphate, coenzyme Q and for prenyl substitutuents on proteins, must be met by intracellular synthesis.
Study of the interactions of lipoproteins with whole cells is very appropriate to the understanding of the functions and metabolism of these macromolecules. Because of their size, lipoproteins could not be expected to enter cells by simple penetration of, or solution in, the plasma membrane. Some form of specific reaction mechanism would seem to be necessary, and indeed this appears to be the case. Cell culture offers many advantages for this type of investigation. Individual cell types can be studied independently from the interactions and compensations which complicate the picture in the whole animal or even in isolated organs. Genetically mutant cells can be compared to their normal counterparts, and this approach has been most fruitful. Obviously, control of the environment is greater in cell culture than in more complex biological systems, and sampling error is lower.
In experimental animals, diets high in polyunsaturated fatty acids (USF) greatly promote tumorigenesis relative to the same animals fed saturated fat (SF)1–6. It appears that the tumor-promoting properties of a high fat diet are more a function of fatty acid composition than of fat content per se or total caloric intake5. Various mechanisms that were examined to explain the promotion of tumorigenesis by USF diet include: alterations in hormone levels, membrane fluidity, intracellular communication, prostaglandins, protein kinases, immune system and cell proliferation5. In spite of studies by cancer biologists and nutritionists, the exact mechanism(s) by which USF diets promote tumorigenesis is not well understood. It is likely that the tumor promoting properties of high USF diets may be related to their capacity to eliminate bile acids, stimulate de novo cholesterogenesis and decrease serum cholesterol ester levels7. Moreover, in various experimental tumor model systems3–5 and in epidemiological studies8, the serum cholesterol and triglyceride levels were found to be reduced. However, a cause and effect relationship between low serum low density lipoproteins (LDL) and promotion of cancer has not been explored thoroughly. Nonetheless, it is widely accepted that there is a cause and effect relationship between serum LDL and coronary heart disease (CHD) and that a reduction in serum LDL will greatly reduce the risk of CHD9.
Some types of cellular regulation are known to be associated with changes in membrane fluidity. For example, it has been shown that the neoplastic transformation of cells from a variety of origins is accompanied by a marked increase in cell surface fluidity (Inbar et
al., 1973; Shinitzky and Inbar, 1974; Ben-Bassat et
al., 1977; Inbar et
al., 1977a). Both cell cycle regulation (deLatt et
al., 1977) and contact inhibition of cells in
vitro (Inbar et
al., 1977b) are also known to be linked with membrane fluidity changes. These changes in fluidity have been demonstrated to be a function of the membrane cholesterol/phospholipid (c/p) ratio (Shinitizky and Inbar, 1974; Chen et
al., 1975; Borochov and Shinitzky, 1976; Cooper et
al., 1978), so that the relatively low ratios are associated with increased fluidity. The c/p ratio in cell membranes can be experimentally manipulated by mixing the cells with liposomes of various cholesterol content (Borochov and Shinitzky, 1976; Cooper et
al., 1978; Inbar and Shinitzky, 1974; Alderson and Green, 1975; Dunnick et
al., 1976; Chen and Keenan, 1977).
Background:
Propionyl-CoA carboxylase (PCC) is a mitochondrial enzyme involved in the catabolism of several essential amino acids and odd chain fatty acids. Previous PCC assays have involved either a radiometric assay or have required mitochondria isolation and/or enzyme purification.
Methods:
We developed an enzymatic method to analyze PCC activity in phytohaemagglutinin (PHA) stimulated lymphocytes that involves high performance liquid chromatography.
Results:
The method shows good linearity and sensitivity. PCC activity was unaffected even when lymphocytes were isolated and PHA stimulated after a whole blood sample had been stored at 4°C for 5days. This indicates that this method is suitable for analyzing samples from distant medical centers. The PCC activity of patients with propionic acidemia was found to be much lower than that of normal individuals and carriers. However, this PCC assay is significantly affected by the red blood cell contamination. In conclusion, this is a reliable method for performing PCC assays and only requires 0.5 to 1.0ml of whole blood from newborns.
Conclusions:
The PCC assay established in this study is useful for the confirmation of PA in individuals, and prenatal diagnosis and genetic counseling for the affected families.
Extensive studies from my laboratory when considered in context with a broad spectrum of work by others, collectively emphasize that an obligatory coupling occurs between the following metabolic phenomena in rapidly growing cells and tumors:
1.
There is an enhanced rate of citrate export from tumor mitochondria, concomitant with a pattern for terminal respiration that is consistent with the operation of a “truncated Krebs cycle”;
2.
There is an enhanced precursor carbon flux (from cytoplasmic citrate or acetyl-CoA) through the tumor’s cholesterogenic pathway, the cause of which appears to depend upon two characteristic enzymatic aberrations: the expression, per tumor cell, of a dramatically increased (and persisting) amount of HMGR, and an equally increased activity of ATP-citrate lyase; and,
3.
A recently discovered set of acidic, isoprenylated (or terpenylated) phospho-proteins arises in proliferating cells (tumor or “normal”), whose synthesis prior to and during the S-phase of the cell cycle mandates a highly active and continuous formation and utilization of cholesterogenic pathway intermediates, especially those generated in the the segment between mevalonic acid and squalene.
The basic role of biomembranes is to provide a barrier between a cell or organelle and its environment and at the same time to serve as a matrix for the association of proteins with lipids (Gibbons et al. 1982) or, as pointed out by Lewis Thomas (1974), “it takes a membrane to make sense out of disorder”. In these membranes, sterols play a major role both architecturally and functionally. The most common membrane sterol in animals is cholesterol (Fig. 1).
Sterols derived from cholesterol by hydroxylation of the side chain in the 20α, 22α, 22β, or 25 position inhibited sterol synthesis from acetate and depressed the level of 3 hydroxy 3 methylglutaryl CoA reductase (EC 1.1.1.34) activity in primary cultures of mouse fetal liver cells and in L cell cultures. Rates of acetate metabolism to fatty acids and CO2, and rates of RNA and protein synthesis were not affected. Following the addition of the most potent inhibitors of the group, 25 hydroxycholesterol and 20α hydroxycholesterol, to L cell cultures the enzyme activity diminished to one half of the original amount within a period of 1 to 1.3 hr. Inhibitory potency was influenced by the location of the hydroxyl function on the side chain, by the completeness of the side chain, and by the introduction of a third functional group into the molecule. (21 references.)
Rates of sterol synthesis from acetate and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMG-CoA reductase, EC 1.1.1.3.4) in liver cell cultures and in L cells were strongly inhibited by highly purified preparations of 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-keto-cholesterol. These steroids specifically diminished the activity of 3-hydroxy-3-methylglutaryl-CoA reductase without altering rates of acetate metabolism to CO2 or fatty acids, or rates of protein and RNA synthesis. The inhibitory activities of these steroids were associated with specific structural features which included the 7-ketone or 7-hydroxyl functions. The following steroids did not inhibit sterol synthesis significantly under the test conditions employed: purified cholesterol, 7-dehydrocholesterol, 24,25-dihydrolanosterol, pregnenolone, cholestane, cholic acid, and chenodeoxycholic acid. 3-Keto-Δ⁴-cholesten-7α-ol inhibited sterol synthesis slightly but did not depress 3-hydroxy-3-methylglutaryl-CoA reductase activity. Other steroid preparations (cholestanol, cholestan-3-one, and cholest-4-en-3-one) appeared to be weakly inhibitory when they were added to liver cell cultures at relatively high concentrations. Sterol synthesis in L cells was more sensitive to inhibitory steroids than was that in liver cells, although primary liver cell cultures took up more [4-¹⁴C]cholesterol from the medium than did L cells or long term liver cell cultures.
Measurements of the inhibitory activities of fractions isolated from impure cholesterol and from a mixture of preputial gland sterols by thin layer chromatography suggest that there are other inhibitors of sterol synthesis not yet identified.
CELLS growing in a chemically defined, sterol-free medium must synthesise cholesterol or, in the case of L cells, desmosterol1 for membrane formation. Studies by others showed that when cholesterol or desmosterol was added to L-cell cultures the exogenous sterol was utilised and the rate of sterol synthesis was diminished, presumably by a negative feedback mechanism2,3. Our observations4, however, provide new information regarding the structures of sterols that affect sterol synthesis. Exogenous cholesterol and various metabolically related steroids do not inhibit sterol synthesis in mouse liver cell or fibroblast cultures under conditions where derivatives of cholesterol oxygenated in the 7, 20, 22 or 25 positions are highly inhibitory. These inhibitory sterols, at concentrations of 0.02-0.2 µg ml-1 specifically depress the activity of the regulatory enzyme in the sterol synthetic pathway, 3-hydroxy-3-methylgrutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), within 6 h.
Cell suspensions of normal and leukemic mouse lymphocytes from spleens, thymuses, and mesenteric lymph nodes actively incorporate acetate into cellular lipids. The de novo synthesis of digitonin precipitable sterols in leukemic cells is tenfold greater than that of normal cells and is associated with a correspondingly increased activity of the rate limiting enzyme, 3 hydroxy 3 methylglutaryl coenzyme A reductase. In contrast, synthesis of fatty acids is only slightly enhanced and production of CO2 is not affected because of leukemogenesis.
SUMMARY Bone marrow cells and peripheral blood leukocytes from leukemic AKR/J mice synthesize sterols from acetate at a vastly greater rate than the respective cells from normal AKR/J mice, i.e., approximately 20- and 100-fold. The controlling enzyme of sterol synthesis, 3-hydroxy-3-melhyl- glutaryl coenzyme A reducÃase,is readily detectable in gradient-purified leukocytes from leukemic mice, whereas normal leukocytes, fractions of red blood cells, and platelets express very low activity. The increased rate of sterol synthesis in leukocytes and bone marrow cells of leukemic animals does not result in an elevated plasma cholesterol level.
A two-step centrifugation technique is described in which 80 to 90 per cent of the total lymphocyte population of human and animal blood can be obtained free of erythrocytes, granulocytes, and platelets. Uncontaminated platelet populations were similarly obtained. The procedure was scaled sufficiently to allow the purification of large numbers of functionally viable cells in a relatively short period of time. The procedure was applied to purification of splenic lymphocytes, and pure lymphocyte populations were obtained with the selective exclusion of nonviable cells.
- L Liljeqvist
- J Gurtler
- R Blomstrad
Liljeqvist, L., Gurtler, J. & Blomstrad, R. (1973) Acta
Chem. Scan. 27, 197-208.
- A A Kandutsch
- H W Chen
Kandutsch, A. A. & Chen, H. W. (1973) J. Biol. Chem. 248,
8408-8417.