4-Octyl Itaconate Alleviates Airway Eosinophilic Inflammation by Suppressing Chemokines and Eosinophil Development.

4-Octyl itaconate (4-OI) is a derivative of the Krebs cycle-derived metabolite itaconate and displays an array of antimicrobial and anti-inflammatory properties through modifying cysteine residues within protein targets. We have found that 4-OI significantly reduces the production of eosinophil-targeted chemokines in a variety of cell types, including M1 and M2 macrophages, Th2 cells, and A549 respiratory epithelial cells. Notably, the suppression of these chemokines in M1 macrophages was found to be NRF2-dependent. In addition, 4-OI can interfere with IL-5 signaling and directly affect eosinophil differentiation. In a model of eosinophilic airway inflammation in BALB/c mice, 4-OI alleviated airway resistance and reduced eosinophil recruitment to the lungs. Our findings suggest that itaconate derivatives could be promising therapeutic agents for the treatment of eosinophilic asthma.

The Circadian Clock Protein BMAL1 Acts as a Metabolic Sensor In Macrophages to Control the Production of Pro IL-1ß.

The transcription factor BMAL1 is a clock protein that generates daily or circadian rhythms in physiological functions including the inflammatory response of macrophages. Intracellular metabolic pathways direct the macrophage inflammatory response, however whether the clock is impacting intracellular metabolism to direct this response is unclear. Specific metabolic reprogramming of macrophages controls the production of the potent pro-inflammatory cytokine IL-1ß. We now describe that the macrophage molecular clock, through Bmal1, regulates the uptake of glucose, its flux through glycolysis and the Krebs cycle, including the production of the metabolite succinate to drive Il-1ß production. We further demonstrate that BMAL1 modulates the level and localisation of the glycolytic enzyme PKM2, which in turn activates STAT3 to further drive Il-1ß mRNA expression. Overall, this work demonstrates that BMAL1 is a key metabolic sensor in macrophages, and its deficiency leads to a metabolic shift of enhanced glycolysis and mitochondrial respiration, leading to a heightened pro-inflammatory state. These data provide insight into the control of macrophage driven inflammation by the molecular clock, and the potential for time-based therapeutics against a range of chronic inflammatory diseases.

Targeting immunometabolism as an anti-inflammatory strategy.

The growing field of immunometabolism has taught us how metabolic cellular reactions and processes not only provide a means to generate ATP and biosynthetic precursors, but are also a way of controlling immunity and inflammation. Metabolic reprogramming of immune cells is essential for both inflammatory as well as anti-inflammatory responses. Four anti-inflammatory therapies, DMF, Metformin, Methotrexate and Rapamycin all work by affecting metabolism and/or regulating or mimicking endogenous metabolites with anti-inflammatory effects. Evidence is emerging for the targeting of specific metabolic events as a strategy to limit inflammation in different contexts. Here we discuss these recent developments and speculate on the prospect of targeting immunometabolism in the effort to develop novel anti-inflammatory therapeutics. As accumulating evidence for roles of an intricate and elaborate network of metabolic processes, including lipid, amino acid and nucleotide metabolism provides key focal points for developing new therapies, we here turn our attention to glycolysis and the TCA cycle to provide examples of how metabolic intermediates and enzymes can provide potential novel therapeutic targets.

Caspase-11 promotes allergic airway inflammation.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.

Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity.

Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate during glycolysis. The PK isoform PKM2 has additional roles in regulation of gene transcription and protein phosphorylation. PKM2 has been shown to control macrophage metabolic remodeling in inflammation, but its role in T cell biology is poorly understood. Here, we report PKM2 upregulation, phosphorylation, and nuclear accumulation in murine and human CD4+ T cells following activation in vitro. Treatment of T cells with TEPP-46, an allosteric activator that induces PKM2 tetramerization and blocks its nuclear translocation, strongly reduces their activation, proliferation, and cytokine production by inhibiting essential signaling pathways and thus preventing the engagement of glycolysis. TEPP-46 limits the development of both T helper 17 (Th17) and Th1 cells in vitro and ameliorates experimental autoimmune encephalomyelitis (EAE) in vivo. Overall, our results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation.

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1ß and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1-/- mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.

Specific Inhibition of the NLRP3 Inflammasome as an Antiinflammatory Strategy in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) pulmonary disease is characterized by chronic infection with Pseudomonas aeruginosa and sustained neutrophil-dominant inflammation. The lack of effective antiinflammatory therapies for people with CF (PWCF) represents a significant challenge.Objectives: To identify altered immunometabolism in the CF neutrophil and investigate the feasibility of specific inhibition of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome as a CF antiinflammatory strategy in vivo.Methods: Key markers of increased aerobic glycolysis, known as a Warburg effect, including cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, succinate, HIF-1α (hypoxia-inducible factor-1α), lactate, and the IL-1ß precursor pro-IL-1ß, as well as caspase-1 activity and processing of pro-IL-1ß to IL-1ß by the NLRP3 inflammasome, were measured in neutrophils from blood and airway secretions from healthy control subjects (n = 12), PWCF (n = 16), and PWCF after double-lung transplantation (n = 6). The effects of specific inhibition of NLRP3 on airway inflammation and bacterial clearance in a murine CF model were subsequently assessed in vivo.Measurements and Main Results: CF neutrophils display increased aerobic glycolysis in the systemic circulation. This effect is driven by low-level endotoxemia, unaffected by CFTR (cystic fibrosis transmembrane conductance regulator) modulation, and resolves after transplant. The increased pro-IL-1ß produced is processed to its mature active form in the LPS-rich CF lung by the NLRP3 inflammasome via caspase-1. Specific NLRP3 inhibition in vivo with MCC950 inhibited IL-1ß in the lungs of CF mice (P < 0.0001), resulting in significantly reduced airway inflammation and improved Pseudomonas clearance (P < 0.0001).Conclusions: CF neutrophil immunometabolism is altered in response to inflammation. NLRP3 inflammasome inhibition may have an antiinflammatory and anti-infective role in CF.

Malonylation of GAPDH is an inflammatory signal in macrophages.

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.

Circadian clock protein BMAL1 regulates IL-1ß in macrophages via NRF2.

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1ß and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1-/- macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1ß. The excessive prooxidant and proinflammatory phenotype of Bmal1-/- macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1ß are known to play a role.

Metabolic Modulation in Macrophage Effector Function.

Traditionally cellular respiration or metabolism has been viewed as catabolic and anabolic pathways generating energy and biosynthetic precursors required for growth and general cellular maintenance. However, growing literature provides evidence of a much broader role for metabolic reactions and processes in controlling immunological effector functions. Much of this research into immunometabolism has focused on macrophages, cells that are central in pro- as well as anti-inflammatory responses-responses that in turn are a direct result of metabolic reprogramming. As we learn more about the precise role of metabolic pathways and pathway intermediates in immune function, a novel opportunity to target immunometabolism therapeutically has emerged. Here, we review the current understanding of the regulation of macrophage function through metabolic remodeling.

Pyruvate Kinase M2 Is Required for the Expression of the Immune Checkpoint PD-L1 in Immune Cells and Tumors.

Blocking interaction of the immune checkpoint receptor PD-1 with its ligand PD-L1 is associated with good clinical outcomes in a broad variety of malignancies. High levels of PD-L1 promote tumor growth by restraining CD8+ T-cell responses against tumors. Limiting PD-L1 expression and function is therefore critical for allowing the development of antitumor immune responses and effective tumor clearance. Pyruvate kinase isoform M2 (PKM2) is also a key player in regulating cancer as well as immune responses. PKM2 catalyzes the final rate-limiting step of glycolysis. Furthermore, PKM2 as a dimer translocates to the nucleus, where it stimulates hypoxia-inducible factor 1α (Hif-1α) transactivation domain function and recruitment of p300 to the hypoxia response elements (HRE) of Hif-1α target genes. Here, we provide the first evidence of a role for PKM2 in regulating the expression of PD-L1 on macrophages, dendritic cells (DCs), T cells, and tumor cells. LPS-induced expression of PD-L1 in primary macrophages was inhibited by the PKM2 targeting compound TEPP-46. Furthermore, RNA silencing of PKM2 inhibited LPS-induced PD-L1 expression. This regulation occurs through direct binding of PKM2 and Hif-1α to HRE sites on the PD-L1 promoter. Moreover, TEPP-46 inhibited expression of PD-L1 on macrophages, DCs, and T cells as well as tumor cells in a mouse CT26 cancer model. These findings broaden our understanding of how PKM2 may contribute to tumor progression and may explain the upregulation of PD-L1 in the tumor microenvironment.

The Induction of Pro-IL-1ß by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production.

PGE2 has been shown to increase the transcription of pro-IL-1ß. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1ß by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE2 on IL-1ß production. We have found that in murine bone marrow-derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1ß transcription, as well as boosting the ability of LPS to induce IL-1ß mRNA and pro-IL-1ß while inhibiting the production of TNF-α. This results in an increase in mature IL-1ß production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1ß production. By blocking PGE2 production with indomethacin, we made a striking finding that endogenous PGE2 is essential for LPS-induced pro-IL-1ß production, suggesting a positive feedback loop. The effect of endogenous PGE2 was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1ß, PGE2 boosted LPS-induced IL-1ß production. PGE2 did not inhibit ATP-induced mature IL-1ß production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1ß, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1ß and back to PGE2, which itself is induced by IL-1ß, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE2 on TNF-α production.

Pyruvate Kinase M2: A Potential Target for Regulating Inflammation.

Pyruvate kinase (PK) is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signaling pathways, affecting both the enzymatic activity of PKM2 as a PK and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for this protein as a therapeutic target in inflammatory disorders.

Cutting Edge: Mycobacterium tuberculosis Induces Aerobic Glycolysis in Human Alveolar Macrophages That Is Required for Control of Intracellular Bacillary Replication.

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1ß, a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis, we performed metabolic and functional studies on human alveolar macrophages, human monocyte-derived macrophages, and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1ß and decreased transcription of PTGS2, increased levels of anti-inflammatory IL-10, and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival, demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1ß. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies.

Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.

The response to an innate immune challenge is conditioned by the time of day, but the molecular basis for this remains unclear. In myeloid cells, there is a temporal regulation to induction by lipopolysaccharide (LPS) of the proinflammatory microRNA miR-155 that correlates inversely with levels of BMAL1. BMAL1 in the myeloid lineage inhibits activation of NF-κB and miR-155 induction and protects mice from LPS-induced sepsis. Bmal1 has two miR-155-binding sites in its 3'-UTR, and, in response to LPS, miR-155 binds to these two target sites, leading to suppression of Bmal1 mRNA and protein in mice and humans. miR-155 deletion perturbs circadian function, gives rise to a shorter circadian day, and ablates the circadian effect on cytokine responses to LPS. Thus, the molecular clock controls miR-155 induction that can repress BMAL1 directly. This leads to an innate immune response that is variably responsive to challenges across the circadian day.

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.

LPS induces the degradation of programmed cell death protein 4 (PDCD4) to release Twist2, activating c-Maf transcription to promote interleukin-10 production.

Programmed cell death protein 4 (PDCD4) is a tumor suppressor and has also been shown to suppress production of the immunomodulatory cytokine IL-10. The precise role of PDCD4 in IL-10 induction in macrophages is still not fully understood. Incubation of macrophages with inhibitors of PI3K and mTOR blocked LPS-stimulated PDCD4 degradation and expression of c-Maf and IL-10 production. PDCD4 and the transcription factor Twist2 were shown to form a complex in untreated cells. LPS disrupted the complex allowing Twist2 to bind to the c-Maf promoter. PI3K and mTOR inhibitors prevented this disruption by stabilizing PDCD4 and thereby decreased Twist2 binding to the c-Maf promoter and induction of c-Maf mRNA. These results indicate a regulatory role for PDCD4 and Twist2 in LPS-induced IL-10 production in macrophages. LPS promotes PDCD4 degradation via a pathway involving PI3K and mTOR, releasing Twist2, which induces IL-10 via c-Maf.

The Warburg effect then and now: from cancer to inflammatory diseases.

Inflammatory immune cells, when activated, display much the same metabolic profile as a glycolytic tumor cell. This involves a shift in metabolism away from oxidative phosphorylation towards aerobic glycolysis, a phenomenon known as the Warburg effect. The result of this change in macrophages is to rapidly provide ATP and metabolic intermediates for the biosynthesis of immune and inflammatory proteins. In addition, a rise in certain tricarboxylic acid cycle intermediates occurs notably in citrate for lipid biosynthesis, and succinate, which activates the transcription factor Hypoxia-inducible factor. In this review we take a look at the emerging evidence for a role for the Warburg effect in the immune and inflammatory responses. The reprogramming of metabolic pathways in macrophages, dendritic cells, and T cells could have relevance in the pathogenesis of inflammatory and metabolic diseases and might provide novel therapeutic strategies.

Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21.

The tumor suppressor PDCD4 is a proinflammatory protein that promotes activation of the transcription factor NF-kappaB and suppresses interleukin 10 (IL-10). Here we found that mice deficient in PDCD4 were protected from lipopolysaccharide (LPS)-induced death. The induction of NF-kappaB and IL-6 by LPS required PDCD4, whereas LPS enhanced IL-10 induction in cells lacking PDCD4. Treatment of human peripheral blood mononuclear cells with LPS resulted in lower PDCD4 expression, which was due to induction of the microRNA miR-21 via the adaptor MyD88 and NF-kappaB. Transfection of cells with a miR-21 precursor blocked NF-kappaB activity and promoted IL-10 production in response to LPS, whereas transfection with antisense oligonucleotides to miR-21 or targeted protection of the miR-21 site in Pdcd4 mRNA had the opposite effect. Thus, miR-21 regulates PDCD4 expression after LPS stimulation.