Alcohol-induced lipid dysregulation impairs glycolytic responses to LPS in alveolar macrophages.
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ABSTRACT:
SUBMITTER: Slovinsky WS
PROVIDER: S-EPMC8094043 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Excessive alcohol use increases the risk of developing respiratory infections partially due to impaired alveolar macrophage (AM) phagocytic capacity. Previously, we showed that chronic ethanol (EtOH) exposure led to mitochondrial derangements and diminished oxidative phosphorylation in AM. Since oxidative phosphorylation is needed to meet the energy demands of phagocytosis, EtOH mediated decreases in oxidative phosphorylation likely contribute to impaired AM phagocytosis. Treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, pioglitazone (PIO), improved EtOH-mediated decreases in oxidative phosphorylation. In other models, hypoxia-inducible factor-1 alpha (HIF-1α) has been shown to mediate the switch from oxidative phosphorylation to glycolysis; however, the role of HIF-1α in chronic EtOH mediated derangements in AM has not been explored. We hypothesize that AM undergo a metabolic shift from oxidative phosphorylation to a glycolytic phenotype in response to chronic EtOH exposure. Further, we speculate that HIF-1α is a critical mediator of this metabolic switch. To test these hypotheses, primary mouse AM (mAM) were isolated from a mouse model of chronic EtOH consumption and a mouse AM cell line (MH-S) were exposed to EtOH in vitro. Expression of HIF-1α, glucose transporters (Glut1 and 4), and components of the glycolytic pathway (Pfkfb3 and PKM2), were measured by qRT-PCR and western blot. Lactate levels (lactate assay), cell energy phenotype (extracellular flux analyzer), glycolysis stress tests (extracellular flux analyzer), and phagocytic function (fluorescent microscopy) were conducted. EtOH exposure increased expression of HIF-1α, Glut1, Glut4, Pfkfb3, and PKM2 and shifted AM to a glycolytic phenotype. Pharmacological stabilization of HIF-1α via cobalt chloride treatment in vitro mimicked EtOH-induced AM derangements (increased glycolysis and diminished phagocytic capacity). Further, PIO treatment diminished HIF-1α levels and reversed glycolytic shift following EtOH exposure. These studies support a critical role for HIF-1α in mediating the glycolytic shift in energy metabolism of AM during excessive alcohol use.
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Project description:There is a strong epidemiological link between smoking and tuberculosis (TB), but the association is confounded by socioeconomic and other factors. A direct relationship between cigarette smoke and poor treatment-related outcomes in patients with TB is therefore questionable. We investigated whether constituents of tobacco smoke impair mycobacterial host immune responses in vitro.Preparation of a cigarette smoke extract (CSE) from Marlboro Red cigarettes was standardised and reproducibility verified by mass spectroscopy. Macrophages were derived from peripheral blood monocytes (MDM) and alveolar macrophages from bronchoalveolar lavage fluid from healthy non-smoking volunteers. Mycobacterial uptake (flow cytometric detection of fluorescence using green fluorescent protein-labelled BCG), cytokine responses (ELISA) and mycobacterial containment (colony forming units) was evaluated in both macrophage populations with and without co-culture with CSE, nicotine and a nicotine receptor blocker.Cigarette smoke failed to impair the uptake of mycobacteria by monocyte-derived or alveolar macrophages. CSE (vs no CSE) reduced the mean (SD) BCG-driven macrophage (MDM) interferon ? (IFN-?), tumour necrosis factor ? (TNF-?) and interleukin 10 (IL-10) responses by 56.4 (18.6)%, 67.0 (33.4)% and 77.7 (27.7)%, respectively (p<0.001). Nicotine alone impaired IL-10 and TNF-? production by 48.8 (37)% and 49 (50)%, respectively (p<0.05) through an ?-7 nicotine receptor-independent mechanism. In 5-day cultures, CSE impaired mycobacterial (BCG) containment in both monocyte-derived and alveolar macrophages.Cigarette smoke attenuates effector cytokine responses and impairs mycobacterial containment within infected human macrophages derived from the peripheral blood and alveolar compartments, thus supporting the hypothesis that cigarette smoke subverts mycobacteria-related immunity.
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Project description:Silicosis is a common occupational disease and represents a significant contributor to respiratory morbidity and mortality worldwide. Lipid-laden macrophages, or foam cells, are observed in the lungs of patients with silicosis but the mechanisms mediating their formation remain poorly understood. In this study, we sought to elucidate the mechanisms by which silica promotes foam cell formation in the lung, and to determine whether uptake of lipids alone is sufficient to drive TGF-β production by alveolar macrophages. Consistent with previous reports, we found that foam cells were markedly increased in the lungs of patients with silicosis and that these findings associated with both higher levels of intracellular lipid levels (oxidized LDL, ox-LDL) and elevated transcript levels for the lipid scavenger receptor CD36 and the nuclear receptor PPARγ. Employing a rat alveolar macrophage cell line, we found that exposure to silica dust or ox-LDL alone had a modest effect on the induction of foam cell formation and only silica was capable of inducing the production of TGF-β. In contrast, foam cell formation and TGF-β production were both dramatically increased when cells were exposed to a combination of silica dust and ox-LDL. Moreover, we found that these endpoints were markedly attenuated by either blocking CD36 or inhibiting the activity of PPARγ. Altogether, our findings suggest that foam cell formation and TGF-β production are driven by the simultaneous uptake of silica and lipids in alveolar macrophages and that strategies aimed at blocking lipid uptake by alveolar macrophages might be effective in ameliorating fibrotic responses to silica in the lung.
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Project description:Macrophage effector function is dynamic in nature and largely dependent on not only the type of immunological challenge but also the tissue-specific environment and developmental origin of a given macrophage population. Recent research has highlighted the importance of glycolytic metabolism in the regulation of effector function as a common feature associated with macrophage activation. Yet, most research has used macrophage cell lines and bone marrow-derived macrophages, which do not account for the diversity of macrophage populations and the role of tissue specificity in macrophage immunometabolism. Tissue-resident alveolar macrophages (TR-AMs) reside in an environment characterized by remarkably low glucose concentrations, making glycolysis-linked immunometabolism an inefficient and unlikely means of immune activation. In this study, we show that TR-AMs rely on oxidative phosphorylation to meet their energy demands and maintain extremely low levels of glycolysis under steady-state conditions. Unlike bone marrow-derived macrophages, TR-AMs did not experience enhanced glycolysis in response to LPS, and glycolytic inhibition had no effect on their proinflammatory cytokine production. Hypoxia-inducible factor 1α stabilization promoted glycolysis in TR-AMs and shifted energy production away from oxidative metabolism at baseline, but it was not sufficient for TR-AMs to mount further increases in glycolysis or enhance immune function in response to LPS. Importantly, we confirmed these findings in an in vivo influenza model in which infiltrating macrophages had significantly higher glycolytic and proinflammatory gene expression than TR-AMs. These findings demonstrate that glycolysis is dispensable for macrophage effector function in TR-AM and highlight the importance of macrophage tissue origin (tissue resident vs. recruited) in immunometabolism.
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Project description:The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been implicated in a variety of diseases, including atherosclerosis, neurodegenerative diseases, and infectious diseases. Thus, inhibitors of NLRP3 inflammasome have emerged as promising approaches to treat inflammation-related diseases. The aim of this study was to explore the effects of juglone (5-hydroxyl-1,4-naphthoquinone) on NLRP3 inflammasome activation. The inhibitory effects of juglone on nitric oxide (NO) production were assessed in lipopolysaccharide (LPS)-stimulated J774.1 cells by Griess assay, while its effects on reactive oxygen species (ROS) and NLRP3 ATPase activity were assessed. The expression levels of NLRP3, caspase-1, and pro-inflammatory cytokines (IL-1β, IL-18) and cytotoxicity of juglone in J774.1 cells were also determined. Juglone was non-toxic in J774.1 cells when used at 10 μM (p < 0.01). Juglone treatment inhibited the production of ROS and NO. The levels of NLRP3 and cleaved caspase-1, as well as the secretion of IL-1β and IL-18, were decreased by treatment with juglone in a concentration-dependent manner. Juglone also inhibited the ATPase activities of NLRP3 in LPS/ATP-stimulated J774.1 macrophages. Our results suggested that juglone could inhibit inflammatory cytokine production and NLRP3 inflammasome activation in macrophages, and should be considered as a therapeutic strategy for inflammation-related diseases.
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Project description:ObjectiveC/EBPβ, a crucial transcription factor, regulates innate immunity and inflammatory responses. However, the role played by C/EBPβ in alveolar macrophage (AM) inflammatory responses remains unknown. This study aimed to investigate the role and mechanism of C/EBPβ in alveolar macrophages (AMs) from the transcriptional level and to search for natural compounds targeting C/EBPβ.MethodsRat AMs were infected with Lv-sh-C/EBPβ and treated with LPS, and the expression levels of iNOS, TNF-α, IL-6, and IL-1β were measured by RT-qPCR, Western blotting, and ELISA. Mechanistically, transcriptome sequencing (RNA-seq) revealed changes in gene expression patterns in AMs after LPS stimulation and C/EBPβ knockdown. Functional enrichment analyses and rescue experiments identified and validated inflammation-associated cell signaling pathways regulated by C/EBPβ. Furthermore, virtual screening was used to search for natural compounds that inhibit C/EBPβ with the structure of helenalin as a reference.ResultsFollowing stimulation with LPS, AMs exhibited an increased expression of C/EBPβ. C/EBPβ knockdown significantly decreased the expression levels of inflammatory mediators. A total of 374 differentially expressed genes (DEGs) were identified between LPS-stimulated C/EBPβ knockdown and negative control cells. The NOD-like receptor signaling may be a key target for C/EBPβ, according to functional enrichment analyses of the DEGs. Further experiments showed that the muramyl dipeptide (MDP, NOD2 agonist) reversed the downregulation of inflammatory mediators and the NF-κB pathway caused by the C/EBPβ knockdown. The virtual screening revealed that N-caffeoyltryptophan, orilotimod, and petasiphenone have comparable pharmacological properties to helenalin (a known C/EBPβ inhibitor) and demonstrate a great binding capacity to C/EBPβ.ConclusionAblation of C/EBPβ may attenuate LPS-induced inflammatory damage in AMs by inhibiting the NOD2 receptor signaling pathway. Three natural compounds, N-caffeoyltryptophan, orilotimod, and petasiphenone, may be potential C/EBPβ inhibitors.
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Project description:The role of specific histone deacetylase (HDAC) proteins in regulating the lipopolysaccharide (LPS)-induced inflammatory response and its underlying mechanisms are unclear. Here, HDAC2, a class I HDAC family protein, is essential for the LPS-triggered inflammatory response in macrophages. LPS stimulation increases HDAC2 expression in macrophages. Knockdown of HDAC2 decreases the expression of proinflammatory genes, such as IL-12, TNF-α and iNOS following stimulation with LPS. The adoptive transfer of HDAC2 knockdown macrophages attenuates the LPS-triggered innate inflammatory response in vivo, and these mice are less sensitive to endotoxin shock and Escherichia coli-induced sepsis. Mechanistically, the c-Jun protein is the main target of HDAC2-mediated LPS-induced production of proinflammatory cytokines. Moreover, HDAC2 knockdown increases the expression of c-Jun, which directly binds the promoters of proinflammatory genes and forms nuclear receptor corepressor complexes to inhibit the transcription of proinflammatory genes in macrophages. These effects are rescued by c-Jun expression. According to the chromatin immunoprecipitation analysis, HDAC2 also selectively suppresses c-Jun expression by directly binding to its promoter and modifying histone acetylation after LPS stimulation. Our findings define a new function and mechanism of the HDAC2/c-Jun signaling network that regulates the LPS-induced immune response in macrophages.
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Project description:Dysregulation of histone deacetylase 6 (HDAC6) can lead to the pathologic states and result in the development of various diseases including cancers and inflammatory diseases. The objective of this study was to elucidate the regulatory role of microRNA-22 (miR-22) in HDAC6-mediated expression of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated macrophages. LPS stimulation induced HDAC6 expression, but suppressed miR-22 expression in macrophages, suggesting possible correlation between HDAC6 and miR-22. Luciferase reporter assays revealed that 3'UTR of HDAC6 was a bona fide target site of miR-22. Transfection of miR-22 mimic significantly inhibited LPS-induced HDAC6 expression, while miR-22 inhibitor further increased LPS-induced HDAC6 expression. LPS-induced activation of NF-?B and AP-1 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. LPS-induced expression of pro-inflammatory cytokines such as TNF-?, IL-1?, and IL-6 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. Taken together, these data provide evidence that miR-22 can downregulate LPS-induced expression of proinflammatory cytokines via suppression of NF-?B and AP-1 axis by targeting HDAC6 in macrophages. [BMB Reports 2020; 53(4): 223-228].
| S-EPMC7196186 | biostudies-literature
Project description:Dexmedetomidine, a highly selective α2-adrenoceptor agonist, has been reported to exert an anti-inflammatory effect in several animal models, but the mechanism remains unclear. Previous studies have shown that hypoxia inducible factor 1α-induced glycolysis is essential for the activation of inflammatory macrophages. However, whether dexmedetomidine influences hypoxia inducible factor 1α-induced glycolysis and thus exerts an anti-inflammatory effect has been poorly investigated. This study aims to elucidate the anti-inflammatory mechanism of dexmedetomidine involving the hypoxia inducible factor 1α-dependent glycolytic pathway. We showed that dexmedetomidine could suppress lipopolysaccharide-induced inflammatory cytokine production; inhibit the extracellular acidification rate, glucose consumption and lactate production; and decrease the expression of glycolytic genes in macrophages. The enhancement of glycolysis by the granulocyte-macrophage colony-stimulating factor or higher concentration of glucose could reverse the anti-inflammatory effect of dexmedetomidine on lipopolysaccharide-treated macrophages. Moreover, dexmedetomidine significantly inhibited the upregulation of hypoxia inducible factor 1α at the mRNA and protein levels. Genetic inhibition of hypoxia inducible factor 1α expression could reverse the anti-inflammatory effect of dexmedetomidine. Taken together, our results indicate that dexmedetomidine attenuates lipopolysaccharide-induced proinflammatory responses partially by suppressing hypoxia inducible factor 1α-dependent glycolysis in macrophages.
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Project description:Macrophages are key immune cells in tissues that are able to adapt their metabolic phenotype in response to different stimuli. Lysine deacetylases are important enzymes for regulating inflammatory gene expression and their inhibitors have anti-inflammatory effects in models of chronic obstructive pulmonary disease (COPD). We hypothesized that these anti-inflammatory effects may be driven by or associated with metabolic changes in macrophages. To validate this hypothesis, we used an unbiased and a targeted proteomic approach to investigate metabolic enzymes and LC- and GC-MS to quantify metabolites in combination with functional parameters in primary murine alveolar-like macrophages after lipopolysaccharide (LPS)-induced activation with and without lysine deacetylase inhibitors. We found that lysine deacetylase inhibitor treatment resulted in reduced production of inflammatory mediators such as TNF-α and IL-1β. However, only minor changes in macrophage metabolism were observed, as only one of the deacetylase inhibitors slightly increased mitochondrial respiration. However, lysine deacetylase inhibition specifically enhanced expression of proteins involved in ubiquitination, and this may be a potential driver of the anti-inflammatory effects of lysine deacetylase inhibitors. Our data illustrate that a multi-omics approach is necessary to gain a better understanding of how macrophages interact with cues from their environment. More detailed insight into the molecular mechanisms of KDAC inhibition is needed and investigating ubiquitination seems a promising next step. This is important as conventional anti-inflammatory drugs like corticosteroids have low effectiveness in many patients and novel therapeutic strategies are urgently needed.
2024-02-21 | PXD040933 | Pride