Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming.

Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase.

Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

Cutting Edge: STING Induces ACLY Activation and Metabolic Adaptations in Human Macrophages through TBK1.

The 2'3'-cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of IFN genes (STING) pathway can sense infection and cellular stress by detecting cytosolic DNA. Upon ligand binding, cGAS produces the cyclic dinucleotide messenger cGAMP, which triggers its receptor STING. Active STING initiates gene transcription through the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB and induces autophagy, but whether STING can cause changes in the metabolism of macrophages is unknown. In this study, we report that STING signaling activates ATP-citrate lyase (ACLY) by phosphorylation in human macrophages. Using genetic and pharmacologic perturbation, we show that STING targets ACLY via its prime downstream signaling effector TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1). We further identify that TBK1 alters cellular metabolism upon cGAMP treatment. Our results suggest that STING-mediated metabolic reprogramming adjusts the cellular response to DNA sensing in addition to transcription factor activation and autophagy induction.

The Single Nucleotide Polymorphism Mal-D96N Mice Provide New Insights into Functionality of Mal in TLR Immune Responses.

MyD88 adaptor-like (Mal) protein is the most polymorphic of the four key adaptor proteins involved in TLR signaling. TLRs play a critical role in the recognition and immune response to pathogens through activation of the prototypic inflammatory transcription factor NF-κB. The study of single nucleotide polymorphisms in TLRs, adaptors, and signaling mediators has provided key insights into the function of the corresponding genes but also into the susceptibility to infectious diseases in humans. In this study, we have analyzed the immune response of mice carrying the human Mal-D96N genetic variation that has previously been proposed to confer protection against septic shock. We have found that Mal-D96N macrophages display reduced cytokine expression in response to TLR4 and TLR2 ligand challenge. Mal-D96N macrophages also display reduced MAPK activation, NF-κB transactivation, and delayed NF-κB nuclear translocation, presumably via delayed kinetics of Mal interaction with MyD88 following LPS stimulation. Importantly, Mal-D96N genetic variation confers a physiological protective phenotype to in vivo models of LPS-, Escherichia coli-, and influenza A virus-induced hyperinflammatory disease in a gene dosage-dependent manner. Together, these results highlight the critical role Mal plays in regulating optimal TLR-induced inflammatory signaling pathways and suggest the potential therapeutic advantages of targeting the Mal D96 signaling nexus.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

Macrophage function in obesity-induced inflammation and insulin resistance.

The steadily increasing obesity epidemic affects currently 30% of western populations and is causative for numerous disorders. It has been demonstrated that immune cells such as macrophages reside in or infiltrate metabolic organs under obese conditions and cause the so-called low-grade inflammation or metaflammation that impairs insulin action thus leading to the development of insulin resistance. Here, we report on data that specifically address macrophage biology/physiology in obesity-induced inflammation and insulin resistance.

Interferons and inflammasomes: Cooperation and counterregulation in disease.

Interferons and the IL-1 family of cytokines have important roles in host defense against invading viruses and bacteria. Inflammasomes, multimeric cytosolic sensors of infection, are required for IL-1ß and IL-18 processing and release. Interferons, IL-1ß, and IL-18 are also implicated in autoimmune disease and chronic inflammation. Although independent but complementary pathways induce these cytokine subsets during infection, in some circumstances the cross-talk between these key inflammatory mediators is a particular requirement for effective host defense. In this review we will summarize recent discoveries concerning the potentiation of inflammasome responses by type I interferons, particularly in patients with gram-negative bacterial infections, and reflect on the molecular mechanisms of IFN-ß's immunosuppressive effects through modulation of inflammasome and IL-1ß signaling in patients with tuberculosis and multiple sclerosis.

ATP-binding and hydrolysis of human NLRP3.

The innate immune system uses inflammasomal proteins to recognize danger signals and fight invading pathogens. NLRP3, a multidomain protein belonging to the family of STAND ATPases, is characterized by its central nucleotide-binding NACHT domain. The incorporation of ATP is thought to correlate with large conformational changes in NLRP3, leading to an active state of the sensory protein. Here we analyze the intrinsic ATP hydrolysis activity of recombinant NLRP3 by reverse phase HPLC. Wild-type NLRP3 appears in two different conformational states that exhibit an approximately fourteen-fold different hydrolysis activity in accordance with an inactive, autoinhibited state and an open, active state. The impact of canonical residues in the nucleotide binding site as the Walker A and B motifs and sensor 1 and 2 is analyzed by site directed mutagenesis. Cellular experiments show that reduced NLRP3 hydrolysis activity correlates with higher ASC specking after inflammation stimulation. Addition of the kinase NEK7 does not change the hydrolysis activity of NLRP3. Our data provide a comprehensive view on the function of conserved residues in the nucleotide-binding site of NLRP3 and the correlation of ATP hydrolysis with inflammasome activity.

SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.

A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

The multifaceted therapeutic value of targeting ATP-citrate lyase in atherosclerosis.

ATP-citrate lyase (Acly) is the target of the new class low-density lipoprotein-cholesterol (LDL-C)-lowering drug bempedoic acid (BA). Acly is a key metabolic enzyme synthesizing acetyl-CoA as the building block of cholesterol and fatty acids. Treatment with BA lowers circulating lipid levels and reduces systemic inflammation, suggesting a dual benefit of this drug for atherosclerosis therapy. Recent studies have shown that targeting Acly in macrophages can attenuate inflammatory responses and decrease atherosclerotic plaque vulnerability. Therefore, it could be beneficial to extend the application of Acly inhibition from solely lipid-lowering by liver-specific inhibition to also targeting macrophages in atherosclerosis. Here, we outline the possibilities of targeting Acly and describe the future needs to translate these findings to the clinic.

1-Deoxysphingolipids cause autophagosome and lysosome accumulation and trigger NLRP3 inflammasome activation.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.Abbreviations: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.

Metabolomic Profiling Reveals Distinct and Mutual Effects of Diet and Inflammation in Shaping Systemic Metabolism in Ldlr-/- Mice.

Changes in modern dietary habits such as consumption of Western-type diets affect physiology on several levels, including metabolism and inflammation. It is currently unclear whether changes in systemic metabolism due to dietary interventions are long-lasting and affect acute inflammatory processes. Here, we investigated how high-fat diet (HFD) feeding altered systemic metabolism and the metabolomic response to inflammatory stimuli. We conducted metabolomic profiling of sera collected from Ldlr-/- mice on either regular chow diet (CD) or HFD, and after an additional low-dose lipopolysaccharide (LPS) challenge. HFD feeding, as well as LPS treatment, elicited pronounced metabolic changes. HFD qualitatively altered the systemic metabolic response to LPS; particularly, serum concentrations of fatty acids and their metabolites varied between LPS-challenged mice on HFD or CD, respectively. To investigate whether systemic metabolic changes were sustained long-term, mice fed HFD were shifted back to CD after four weeks (HFD > CD). When shifted back to CD, serum metabolites returned to baseline levels, and so did the response to LPS. Our results imply that systemic metabolism rapidly adapts to dietary changes. The profound systemic metabolic rewiring observed in response to diet might affect immune cell reprogramming and inflammatory responses.

Pyruvate kinase M2 regulates Hif-1α activity and IL-1ß induction and is a critical determinant of the warburg effect in LPS-activated macrophages.

Macrophages activated by the TLR4 agonist LPS undergo dramatic changes in their metabolic activity. We here show that LPS induces expression of the key metabolic regulator Pyruvate Kinase M2 (PKM2). Activation of PKM2 using two well-characterized small molecules, DASA-58 and TEPP-46, inhibited LPS-induced Hif-1α and IL-1ß, as well as the expression of a range of other Hif-1α-dependent genes. Activation of PKM2 attenuated an LPS-induced proinflammatory M1 macrophage phenotype while promoting traits typical of an M2 macrophage. We show that LPS-induced PKM2 enters into a complex with Hif-1α, which can directly bind to the IL-1ß promoter, an event that is inhibited by activation of PKM2. Both compounds inhibited LPS-induced glycolytic reprogramming and succinate production. Finally, activation of PKM2 by TEPP-46 in vivo inhibited LPS and Salmonella typhimurium-induced IL-1ß production, while boosting production of IL-10. PKM2 is therefore a critical determinant of macrophage activation by LPS, promoting the inflammatory response.