Dietary dicarboxylic acids provide a non-storable alternative fat source that protects mice against obesity.

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

Lipoxin A4 yields an electrophilic 15-oxo metabolite that mediates FPR2 receptor-independent anti-inflammatory signaling.

The enzymatic oxidation of arachidonic acid is proposed to yield trihydroxytetraene species (termed lipoxins) that resolve inflammation via ligand activation of the formyl peptide receptor, FPR2. While cell and murine models activate signaling responses to synthetic lipoxins, primarily 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (lipoxin A4, LXA4), there are expanding concerns about the biological formation, detection and signaling mechanisms ascribed to LXA4 and related di- and tri-hydroxy ω-6 and ω-3 fatty acids. Herein, the generation and actions of LXA4 and its primary 15-oxo metabolite were assessed in control, LPS-activated and arachidonic acid supplemented RAW 264.7 macrophages. Despite protein expression of all enzymes required for LXA4 synthesis, both LXA4 and its 15-oxo-LXA4 metabolite were undetectable. Moreover, synthetic LXA4 and the membrane permeable 15-oxo-LXA4 methyl ester that is rapidly de-esterified to 15-oxo-LXA4, displayed no ligand activity for the putative LXA4 receptor FPR2, as opposed to the FPR2 ligand WKYMVm. Alternatively, 15-oxo-LXA4, an electrophilic α,ß-unsaturated ketone, alkylates nucleophilic amino acids such as cysteine to modulate redox-sensitive transcriptional regulatory protein and enzyme function. 15-oxo-LXA4 activated nuclear factor (erythroid related factor 2)-like 2 (Nrf2)-regulated gene expression of anti-inflammatory and repair genes and inhibited nuclear factor (NF)-κB-regulated pro-inflammatory mediator expression. LXA4 did not impact these macrophage anti-inflammatory and repair responses. In summary, these data show an absence of macrophage LXA4 formation and receptor-mediated signaling actions. Rather, if LXA4 were present in sufficient concentrations, this, and other more abundant mono- and poly-hydroxylated unsaturated fatty acids can be readily oxidized to electrophilic α,ß-unsaturated ketone products that modulate the redox-sensitive cysteine proteome via G-protein coupled receptor-independent mechanisms.

Alternative splice variants of the mitochondrial fission protein DNM1L/Drp1 regulate mitochondrial dynamics and tumor progression in ovarian cancer.

Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Intestinal microbial circadian rhythms drive sex differences in host immunity and metabolism.

Circadian rhythms dynamically regulate sex differences in metabolism and immunity, and circadian disruption increases the risk of metabolic disorders. We investigated the role of sex-specific intestinal microbial circadian rhythms in host metabolism using germ-free and conventionalized mice and manipulation of dietary-derived fat, fiber, and microbiota-accessible carbohydrates. Our findings demonstrate that sex differences in circadian rhythms of genes involved in immunity and metabolism depend on oscillations in microbiota, microbial metabolic functions, and microbial metabolites. Further, we show that consuming an obesogenic, high-fat, low-fiber diet produced sex-specific changes in circadian rhythms in microbiota, metabolites, and host gene expression, which were linked to sex differences in the severity of metabolic dysfunction. Our results reveal that microbial circadian rhythms contribute to sex differences in immunity and metabolism and that dietary factors can entrain new circadian rhythms and modify the magnitude of sex differences in host-microbe circadian dynamics.

The gut protist Tritrichomonas arnold restrains virus-mediated loss of oral tolerance by modulating dietary antigen-presenting dendritic cells.

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.

Meta-omics profiling of the gut-lung axis illuminates metabolic networks and host-microbial interactions associated with elevated lung elastance in a murine model of obese allergic asthma.

Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.

Serum metabolomic signatures of fatty acid oxidation defects differentiate host-response subphenotypes of acute respiratory distress syndrome.

BACKGROUND: Fatty acid oxidation (FAO) defects have been implicated in experimental models of acute lung injury and associated with poor outcomes in critical illness. In this study, we examined acylcarnitine profiles and 3-methylhistidine as markers of FAO defects and skeletal muscle catabolism, respectively, in patients with acute respiratory failure. We determined whether these metabolites were associated with host-response ARDS subphenotypes, inflammatory biomarkers, and clinical outcomes in acute respiratory failure. METHODS: In a nested case-control cohort study, we performed targeted analysis of serum metabolites of patients intubated for airway protection (airway controls), Class 1 (hypoinflammatory), and Class 2 (hyperinflammatory) ARDS patients (N = 50 per group) during early initiation of mechanical ventilation. Relative amounts were quantified by liquid chromatography high resolution mass spectrometry using isotope-labeled standards and analyzed with plasma biomarkers and clinical data. RESULTS: Of the acylcarnitines analyzed, octanoylcarnitine levels were twofold increased in Class 2 ARDS relative to Class 1 ARDS or airway controls (P = 0.0004 and < 0.0001, respectively) and was positively associated with Class 2 by quantile g-computation analysis (P = 0.004). In addition, acetylcarnitine and 3-methylhistidine were increased in Class 2 relative to Class 1 and positively correlated with inflammatory biomarkers. In all patients within the study with acute respiratory failure, increased 3-methylhistidine was observed in non-survivors at 30 days (P = 0.0018), while octanoylcarnitine was increased in patients requiring vasopressor support but not in non-survivors (P = 0.0001 and P = 0.28, respectively). CONCLUSIONS: This study demonstrates that increased levels of acetylcarnitine, octanoylcarnitine, and 3-methylhistidine distinguish Class 2 from Class 1 ARDS patients and airway controls. Octanoylcarnitine and 3-methylhistidine were associated with poor outcomes in patients with acute respiratory failure across the cohort independent of etiology or host-response subphenotype. These findings suggest a role for serum metabolites as biomarkers in ARDS and poor outcomes in critically ill patients early in the clinical course.

Targeting mitochondrial energetics reverses panobinostat- and marizomib-induced resistance in pediatric and adult high-grade gliomas.

In previous studies, we demonstrated that panobinostat, a histone deacetylase inhibitor, and bortezomib, a proteasomal inhibitor, displayed synergistic therapeutic activity against pediatric and adult high-grade gliomas. Despite the remarkable initial response to this combination, resistance emerged. Here, in this study, we aimed to investigate the molecular mechanisms underlying the anticancer effects of panobinostat and marizomib, a brain-penetrant proteasomal inhibitor, and the potential for exploitable vulnerabilities associated with acquired resistance. RNA sequencing followed by gene set enrichment analysis (GSEA) was employed to compare the molecular signatures enriched in resistant compared with drug-naïve cells. The levels of adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD)+ content, hexokinase activity, and tricarboxylic acid (TCA) cycle metabolites required for oxidative phosphorylation to meet their bioenergetic needs were analyzed. Here, we report that panobinostat and marizomib significantly depleted ATP and NAD+ content, increased mitochondrial permeability and reactive oxygen species generation, and promoted apoptosis in pediatric and adult glioma cell lines at initial treatment. However, resistant cells exhibited increased levels of TCA cycle metabolites, which required for oxidative phosphorylation to meet their bioenergetic needs. Therefore, we targeted glycolysis and the electron transport chain (ETC) with small molecule inhibitors, which displayed substantial efficacy, suggesting that resistant cell survival is dependent on glycolytic and ETC complexes. To verify these observations in vivo, lonidamine, an inhibitor of glycolysis and mitochondrial function, was chosen. We produced two diffuse intrinsic pontine glioma (DIPG) models, and lonidamine treatment significantly increased median survival in both models, with particularly dramatic effects in panobinostat- and marizomib-resistant cells. These data provide new insights into mechanisms of treatment resistance in gliomas.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Tet2 deficiency drives liver microbiome dysbiosis triggering Tc1 cell autoimmune hepatitis.

The triggers that drive interferon-γ (IFNγ)-producing CD8 T cell (Tc1 cell)-mediated autoimmune hepatitis (AIH) remain obscure. Here, we show that lack of hematopoietic Tet methylcytosine dioxygenase 2 (Tet2), an epigenetic regulator associated with autoimmunity, results in the development of microbiota-dependent AIH-like pathology, accompanied by hepatic enrichment of aryl hydrocarbon receptor (AhR) ligand-producing pathobionts and rampant Tc1 cell immunity. We report that AIH-like disease development is dependent on both IFNγ and AhR signaling, as blocking either reverts ongoing AIH-like pathology. Illustrating the critical role of AhR-ligand-producing pathobionts in this condition, hepatic translocation of the AhR ligand indole-3-aldehyde (I3A)-releasing Lactobacillus reuteri is sufficient to trigger AIH-like pathology. Finally, we demonstrate that I3A is required for L. reuteri-induced Tc1 cell differentiation in vitro and AIH-like pathology in vivo, both of which are restrained by Tet2 within CD8 T cells. This AIH-disease model may contribute to the development of therapeutics to alleviate AIH.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

Elevated microglial oxidative phosphorylation and phagocytosis stimulate post-stroke brain remodeling and cognitive function recovery in mice.

New research shows that disease-associated microglia in neurodegenerative brains present features of elevated phagocytosis, lysosomal functions, and lipid metabolism, which benefit brain repair. The underlying mechanisms remain poorly understood. Intracellular pH (pHi) is important for regulating aerobic glycolysis in microglia, where Na/H exchanger (NHE1) is a key pH regulator by extruding H+ in exchange of Na+ influx. We report here that post-stroke Cx3cr1-CreER+/-;Nhe1flox/flox (Nhe1 cKO) brains displayed stimulation of microglial transcriptomes of rate-limiting enzyme genes for glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. The other upregulated genes included genes for phagocytosis and LXR/RXR pathway activation as well as the disease-associated microglia hallmark genes (Apoe, Trem2, Spp1). The cKO microglia exhibited increased oxidative phosphorylation capacity, and higher phagocytic activity, which likely played a role in enhanced synaptic stripping and remodeling, oligodendrogenesis, and remyelination. This study reveals that genetic blockade of microglial NHE1 stimulated oxidative phosphorylation immunometabolism, and boosted phagocytosis function which is associated with tissue remodeling and post-stroke cognitive function recovery.

A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells.

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Nitroalkene fatty acids modulate bile acid metabolism and lung function in obese asthma.

Bile acid profiles are altered in obese individuals with asthma. Thus, we sought to better understand how obesity-related systemic changes contribute to lung pathophysiology. We also test the therapeutic potential of nitro-oleic acid (NO2-OA), a regulator of metabolic and inflammatory signaling pathways, to mitigate allergen and obesity-induced lung function decline in a murine model of asthma. Bile acids were measured in the plasma of healthy subjects and individuals with asthma and serum and lung tissue of mice with and without allergic airway disease (AAD). Lung function, indices of inflammation and hepatic bile acid enzyme expression were measured in obese mice with house dust mite-induced AAD treated with vehicle or NO2-OA. Serum levels of glycocholic acid and glycoursodeoxycholic acid clinically correlate with body mass index and airway hyperreactivity whereas murine levels of ß-muricholic acid and tauro-ß-muricholic acid were significantly increased and positively correlated with impaired lung function in obese mice with AAD. NO2-OA reduced murine bile acid levels by modulating hepatic expression of bile acid synthesis enzymes, with a concomitant reduction in small airway resistance and tissue elastance. Bile acids correlate to body mass index and lung function decline and the signaling actions of nitroalkenes can limit AAD by modulating bile acid metabolism, revealing a potential pharmacologic approach to improving the current standard of care.

Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions.

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae-contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under the control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is expressed in vitro only under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse-phase chromatography column (RPCC) and showed that the virulence-activating molecules were retained on the RPCC column and recovered in the eluate. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to noninducing medium and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that the ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regionally specific bile acid species in the intestine.

Metabolic Adaptation of Macrophages as Mechanism of Defense against Crystalline Silica.

Silicosis is a lethal pneumoconiosis for which no therapy is available. Silicosis is a global threat, and more than 2.2 million people per year are exposed to silica in the United States. The initial response to silica is mediated by innate immunity. Phagocytosis of silica particles by macrophages is followed by recruitment of mitochondria to phagosomes, generation of mitochondrial reactive oxygen species, and cytokine (IL-1ß, TNF-α, IFN-ß) release. In contrast with LPS, the metabolic remodeling of silica-exposed macrophages is unclear. This study contrasts mitochondrial and metabolic alterations induced by LPS and silica on macrophages and correlates them with macrophage viability and cytokine production, which are central to the pathogenesis of silicosis. Using high-resolution respirometer and liquid chromatography-high-resolution mass spectrometry, we determined the effects of silica and LPS on mitochondrial respiration and determined changes in central carbon metabolism of murine macrophage cell lines RAW 264.7 and IC-21. We show that silica induces metabolic reprogramming of macrophages. Silica, as well as LPS, enhances glucose uptake and increases aerobic glycolysis in macrophages. In contrast with LPS, silica affects mitochondria respiration, reducing complex I and enhancing complex II activity, to sustain cell viability. These mitochondrial alterations are associated in silica, but not in LPS-exposed macrophages, with reductions of tricarboxylic acid cycle intermediates, including succinate, itaconate, glutamate, and glutamine. Furthermore, in contrast with LPS, these silica-induced metabolic adaptations do not correlate with IL-1ß or TNF-α production, but with the suppressed release of IFN-ß. Our data highlight the importance of complex II activity and tricarboxylic acid cycle remodeling to macrophage survival and cytokine-mediated inflammation in silicosis.