Zbigniew Zasłona

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

The Induction of Pro–IL-1β by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production

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

Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease.

The transcription factor BMAL1 is a core component of the molecular clock, regulating biological pathways that drive 24 h (circadian) rhythms in behaviour and physiology. The molecular clock has a profound influence on innate immune function, and circadian disruption is linked with increased incidence of multiple sclerosis (MS). However, the mechanisms underlying this association are unknown. Here we show that BMAL1 and time-of-day regulate the accumulation and activation of various immune cells in a CNS autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). In myeloid cells, BMAL1 maintains anti-inflammatory responses and reduces T cell polarization. Loss of myeloid BMAL1 or midday immunizations to induce EAE create an inflammatory environment in the CNS through expansion and infiltration of IL-1β-secreting CD11b+Ly6Chi monocytes, resulting in increased pathogenic IL-17+/IFN-γ+ T cells. These findings demonstrate the importance of the molecular clock in modulating innate and adaptive immune crosstalk under autoimmune conditions.Circadian controls of immune responses by the molecular clock have been reported, but the underlying mechanisms are unclear. Here the authors show that the master circadian gene, Bmal1, is essential for modulating the homeostasis of myeloid cells to control pro-inflammatory IL-17+/IFN-γ+ T cells in autoimmunity.

MicroRNA-21 Limits Uptake of Listeria monocytogenes by Macrophages to Reduce the Intracellular Niche and Control Infection

MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21−/− macrophages display an increased bacterial burden with Listeria at 30 min and 2 h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21−/− macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21−/− BMDMs in response to LPS or Listeria. TNF-α was increased in miR-21−/− BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21−/− cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 min), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21−/− macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21−/− BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21−/− mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes.

The circadian protein BMAL1 in myeloid cells is a negative regulator of allergic asthma

Our body clock drives rhythms in the expression of genes that have a 24-h periodicity. The transcription factor BMAL1 is a crucial component of the molecular clock. A number of physiological processes, including immune function, are modulated by the circadian clock. Asthma, a disease with very strong clinical evidence demonstrating regulation by circadian variation, is of particular relevance to circadian control of immunity. Airway hypersensitivity and asthma attacks are more common at night in humans. The molecular basis for this is unknown, and there is no model of asthma in animals with genetic distortion of the molecular clock. We used mice lacking BMAL1 in myeloid cells (BMAL1-LysM-/-) to determine the role of BMAL1 in allergic asthma. Using the ovalbumin model of allergic asthma, we demonstrated markedly increased asthma features, such as increased lung inflammation, demonstrated by drastically higher numbers of eosinophils and increased IL-5 levels in the lung and serum, in BMAL1-LysM-/- mice. In vitro studies demonstrated increased proinflammatory chemokine and mannose receptor expression in IL-4- as well as LPS-treated macrophages from BMAL1-LysM-/- mice compared with wild-type controls. This suggests that Bmal1 is a potent negative regulator in myeloid cells in the context of allergic asthma. Our findings might explain the increase in asthma incidents during the night, when BMAL1 expression is low.

Impaired Production and Diurnal Regulation of Vascular RvDn-3 DPA Increase Systemic Inflammation and Cardiovascular DiseaseNovelty and Significance

Rationale: Diurnal mechanisms are central to regulating host responses. Recent studies uncovered a novel family of mediators termed as specialized proresolving mediators that terminate inflammation without interfering with the immune response. Objective: Herein, we investigated the diurnal regulation of specialized proresolving mediators in humans and their role in controlling peripheral blood leukocyte and platelet activation. Methods and Results: Using lipid mediator profiling and healthy volunteers, we found that plasma concentrations of n-3 docosapentaenoic acid-derived D-series resolvins (RvDn-3 DPA) were regulated in a diurnal manner. The production and regulation of these mediators was markedly altered in patients at risk of myocardial infarct. These changes were associated with decreased 5-lipoxygenase expression and activity, as well as increased systemic adenosine concentrations. We also found a significant negative correlation between plasma RvDn-3 DPA and markers of platelet, monocyte, and neutrophil activation, including CD63 and CD11b. Incubation of RvDn-3 DPA with peripheral blood from healthy volunteers and patients with cardiovascular disease significantly and dose-dependently decreased platelet and leukocyte activation. Furthermore, administration of RvD5n-3 DPA to ApoE−/− (apolipoprotein E deficient) mice significantly reduced platelet–leukocyte aggregates, vascular thromboxane B2 concentrations, and aortic lesions. Conclusions: These results demonstrate that peripheral blood RvDn-3 DPA are diurnally regulated in humans, and dysregulation in the production of these mediators may lead to cardiovascular disease.

Macrophages Remember Cheeseburgers and Promote Inflammation via NLRP3

In a recent study, Christ and colleagues identify a key role for NLRP3/IL-1beta in the induction of innate immune memory in monocytes by the Western diet, promoting atherosclerosis and inflammatory diseases.

The circadian clock protein BMAL1 regulates IL-1β in macrophages via NRF2

Significance The molecular clock provides an anticipatory mechanism, allowing organisms to prepare and respond to daily changes in the external environment. The response of the innate immune system to pathogenic threats is dependent on time of day; however, the molecular mechanisms underlying this have yet to be fully uncovered. We observe that the core molecular clock component, BMAL1, is crucial in promoting an antioxidant response in myeloid cells. Deletion of Bmal1 in macrophages disrupts NRF2 activity, facilitating accumulation of reactive oxygen species and the proinflammatory cytokine, IL-1β. Thus the molecular clock directly controls NRF2 transcriptional activity and antioxidant capacity to regulate IL-1β in myeloid cells. 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-1β 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-1β. 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-1β are known to play a role.

An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75s inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation.