Unlocking potential: the role of the electron transport chain in immunometabolism.

The electron transport chain (ETC) couples electron transfer with proton pumping to generate ATP and it also regulates particular innate and adaptive immune cell function. While NLRP3 inflammasome activation was initially linked to reactive oxygen species (ROS) produced from Complexes I and III, recent research suggests that an intact ETC fueling ATP is needed. Complex II may be responsible for Th1 cell proliferation and in some cases, effector cytokine production. Complex III is required for regulatory T (Treg) cell function, while oxidative phosphorylation (OXPHOS) and Complexes I, IV, and V sustain proliferation and antibody production in B lymphocytes, with OXPHOS also being required for B regulatory (Breg) cell function. Despite challenges, the ETC shows therapeutic targeting potential for immune-related diseases and in immuno-oncology.

Metabolic regulation of type I interferon production.

Over the past decade, there has been a surge in discoveries of how metabolic pathways regulate immune cell function in health and disease, establishing the field of immunometabolism. Specifically, pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, and those involving lipid metabolism have been implicated in regulating immune cell function. Viral infections cause immunometabolic changes which lead to antiviral immunity, but little is known about how metabolic changes regulate interferon responses. Interferons are critical cytokines in host defense, rapidly induced upon pathogen recognition, but are also involved in autoimmune diseases. This review summarizes how metabolic change impacts interferon production. We describe how glycolysis, lipid metabolism (specifically involving eicosanoids and cholesterol), and the TCA cycle-linked intermediates itaconate and fumarate impact type I interferons. Targeting these metabolic changes presents new therapeutic possibilities to modulate type I interferons during host defense or autoimmune disorders.

Itaconate boosts malaria via induction of PD-L1.

The Krebs-cycle-derived metabolite itaconate has been shown to be immunomodulatory, targeting multiple processes in macrophages. Ramalho et al. reveal an additional role for itaconate in malaria.1Plasmodium Chabaudi induces itaconate in dendritic cells (DCs), leading to programmed death-ligand 1 (PD-L1) induction. This suppresses CD8+ T cells, important for host defense against malaria, thereby promoting parasitemia.

A break in mitochondrial endosymbiosis as a basis for inflammatory diseases.

Mitochondria retain bacterial traits due to their endosymbiotic origin, but host cells do not recognize them as foreign because the organelles are sequestered. However, the regulated release of mitochondrial factors into the cytosol can trigger cell death, innate immunity and inflammation. This selective breakdown in the 2-billion-year-old endosymbiotic relationship enables mitochondria to act as intracellular signalling hubs. Mitochondrial signals include proteins, nucleic acids, phospholipids, metabolites and reactive oxygen species, which have many modes of release from mitochondria, and of decoding in the cytosol and nucleus. Because these mitochondrial signals probably contribute to the homeostatic role of inflammation, dysregulation of these processes may lead to autoimmune and inflammatory diseases. A potential reason for the increased incidence of these diseases may be changes in mitochondrial function and signalling in response to such recent phenomena as obesity, dietary changes and other environmental factors. Focusing on the mixed heritage of mitochondria therefore leads to predictions for future insights, research paths and therapeutic opportunities. Thus, whereas mitochondria can be considered 'the enemy within' the cell, evolution has used this strained relationship in intriguing ways, with increasing evidence pointing to the recent failure of endosymbiosis being critical for the pathogenesis of inflammatory diseases.

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.

Characterization and inhibition of inflammasome responses in severe and non-severe asthma.

BACKGROUND: Increased airway NLRP3 inflammasome-mediated IL-1ß responses may underpin severe neutrophilic asthma. However, whether increased inflammasome activation is unique to severe asthma, is a common feature of immune cells in all inflammatory types of severe asthma, and whether inflammasome activation can be therapeutically targeted in patients, remains unknown. OBJECTIVE: To investigate the activation and inhibition of inflammasome-mediated IL-1ß responses in immune cells from patients with asthma. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with non-severe (n = 59) and severe (n = 36 stable, n = 17 exacerbating) asthma and healthy subjects (n = 39). PBMCs were stimulated with nigericin or lipopolysaccharide (LPS) alone, or in combination (LPS + nigericin), with or without the NLRP3 inhibitor MCC950, and the effects on IL-1ß release were assessed. RESULTS: PBMCs from patients with non-severe or severe asthma produced more IL-1ß in response to nigericin than those from healthy subjects. PBMCs from patients with severe asthma released more IL-1ß in response to LPS + nigericin than those from non-severe asthma. Inflammasome-induced IL-1ß release from PBMCs from patients with severe asthma was not increased during exacerbation compared to when stable. Inflammasome-induced IL-1ß release was not different between male and female, or obese and non-obese patients and correlated with eosinophil and neutrophil numbers in the airways. MCC950 effectively suppressed LPS-, nigericin-, and LPS + nigericin-induced IL-1ß release from PBMCs from all groups. CONCLUSION: An increased ability for inflammasome priming and/or activation is a common feature of systemic immune cells in both severe and non-severe asthma, highlighting inflammasome inhibition as a universal therapy for different subtypes of disease.

C5aR2 Regulates STING-Mediated Interferon Beta Production in Human Macrophages.

The complement system mediates diverse regulatory immunological functions. C5aR2, an enigmatic receptor for anaphylatoxin C5a, has been shown to modulate PRR-dependent pro-inflammatory cytokine secretion in human macrophages. However, the specific downstream targets and underlying molecular mechanisms are less clear. In this study, CRISPR-Cas9 was used to generate macrophage models lacking C5aR2, which were used to probe the role of C5aR2 in the context of PRR stimulation. cGAS and STING-induced IFN-ß secretion was significantly increased in C5aR2 KO THP-1 cells and C5aR2-edited primary human monocyte-derived macrophages, and STING and IRF3 expression were increased, albeit not significantly, in C5aR2 KO cell lines implicating C5aR2 as a regulator of the IFN-ß response to cGAS-STING pathway activation. Transcriptomic analysis by RNAseq revealed that nucleic acid sensing and antiviral signalling pathways were significantly up-regulated in C5aR2 KO THP-1 cells. Altogether, these data suggest a link between C5aR2 and nucleic acid sensing in human macrophages. With further characterisation, this relationship may yield therapeutic options in interferon-related pathologies.

Fumarate hydratase as a metabolic regulator of immunity.

Tricarboxylic acid (TCA) cycle metabolites have been implicated in modulating signalling pathways in immune cells. Notable examples include succinate and itaconate, which have pro- and anti-inflammatory roles, respectively. Recently, fumarate has emerged as having specific roles in macrophage activation, regulating the production of such cytokines as interleukin (IL)-10 and type I interferons (IFNs). Fumarate hydratase (FH) has been identified as a control point. Notably, FH loss in different models and cell types has been found to lead to DNA and RNA release from mitochondria which are sensed by cytosolic nucleic acid sensors including retinoic acid-inducible gene (RIG)-I, melanoma differentiation-associated protein (MDA)5, and cyclic GMP-AMP synthase (cGAS) to upregulate IFN-ß production. These findings may have relevance in the pathogenesis and treatment of diseases associated with decreased FH levels such as systemic lupus erythematosus (SLE) or FH-deficient kidney cancer.

4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1.

IMPORTANCE: Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics.

4-Octyl Itaconate and Dimethyl Fumarate Induce Secretion of the Anti-Inflammatory Protein Annexin A1 via NRF2.

Annexin A1 is a key anti-inflammatory effector protein that is involved in the anti-inflammatory effects of glucocorticoids. 4-Octyl itaconate (4-OI), a derivative of the endogenous metabolite itaconate, which is abundantly produced by LPS-activated macrophages, has recently been identified as a potent anti-inflammatory agent. The anti-inflammatory effects of 4-OI share a significant overlap with those of dimethyl fumarate (DMF), a derivate of another Krebs cycle metabolite fumarate, which is already in use clinically for the treatment of inflammatory diseases. In this study we show that both 4-OI and DMF induce secretion of the 33-kDa form of annexin A1 from murine bone marrow-derived macrophages, an effect that is much more pronounced in LPS-stimulated cells. We also show that this 4-OI- and DMF-driven annexin A1 secretion is NRF2-dependent and that other means of activating NRF2 give rise to the same response. Lastly, we demonstrate that the cholesterol transporter ABCA1, which has previously been implicated in annexin A1 secretion, is required for this process in macrophages. Our findings contribute to the growing body of knowledge on the anti-inflammatory effects of the Krebs cycle metabolite derivatives 4-OI and DMF.

NLRP12 drives PANoptosis in response to heme.

PANoptosomes are large cell death-inducing complexes that drive a type of cell death called PANoptosis during infection and inflammation. Sundaram and colleagues recently identified NLRP12 as a PANoptosome that induces PANoptosis in response to heme, TNF, and pathogen-associated molecular patterns (PAMPs), indicating a role for NLRP12 in hemolytic and inflammatory diseases.

Dimethyl fumarate and 4-octyl itaconate are anticoagulants that suppress Tissue Factor in macrophages via inhibition of Type I Interferon.

Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.

Immunometabolism at the crossroads of obesity and cancer-a Keystone Symposia report.

Immunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity.

An Emerging Role for Type I Interferons as Critical Regulators of Blood Coagulation.

Type I interferons (IFNs) are central mediators of anti-viral and anti-bacterial host defence. Detection of microbes by innate immune cells via pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and cGAS-STING, induces the expression of type I IFN-stimulated genes. Primarily comprising the cytokines IFN-α and IFN-ß, type I IFNs act via the type I IFN receptor in an autocrine or exocrine manner to orchestrate rapid and diverse innate immune responses. Growing evidence pinpoints type I IFN signalling as a fulcrum that not only induces blood coagulation as a core feature of the inflammatory response but is also activated by components of the coagulation cascade. In this review, we describe in detail recent studies identifying the type I IFN pathway as a modulator of vascular function and thrombosis. In addition, we profile discoveries showing that thrombin signalling via protease-activated receptors (PARs), which can synergize with TLRs, regulates the host response to infection via induction of type I IFN signalling. Thus, type I IFNs can have both protective (via maintenance of haemostasis) and pathological (facilitating thrombosis) effects on inflammation and coagulation signalling. These can manifest as an increased risk of thrombotic complications in infection and in type I interferonopathies such as systemic lupus erythematosus (SLE) and STING-associated vasculopathy with onset in infancy (SAVI). We also consider the effects on coagulation of recombinant type I IFN therapies in the clinic and discuss pharmacological regulation of type I IFN signalling as a potential mechanism by which aberrant coagulation and thrombosis may be treated therapeutically.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

NLRP3, the inflammasome and COVID-19 infection.

Severe coronavirus disease 2019 (COVID-19) is characterized by respiratory failure, shock or multiorgan dysfunction, often accompanied by systemic hyperinflammation and dysregulated cytokine release. These features are linked to the intense and rapid stimulation of the innate immune response. The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a central player in inflammatory macrophage activation which via caspase-1 activation leads to the release of the mature forms of the proinflammatory cytokines interleukin (IL)-1ß and IL-18, and via cleavage of Gasdermin D pyroptosis, an inflammatory form of cell death. Here, we discuss the role of NLRP3 activation in COVID-19 and clinical trials currently underway to target NLRP3 to treat severe COVID-19.

Protein targeting by the itaconate family in immunity and inflammation.

Immune cells are metabolically plastic and respond to inflammatory stimuli with large shifts in metabolism. Itaconate is one of the most up-regulated metabolites in macrophages in response to the gram negative bacterial product LPS. As such, itaconate has recently been the subject of intense research interest. The artificial derivatives, including 4-Octyl Itaconate (4-OI) and Dimethyl Itaconate (DI) and naturally produced isomers, mesaconate and citraconate, have been tested in relation to itaconate biology with similarities and differences in the biochemistry and immunomodulatory properties of this family of compounds emerging. Both itaconate and 4-OI have been shown to modify cysteines on a range of target proteins, with the modification being linked to a functional change. Targets include KEAP1 (the NRF2 inhibitor), GAPDH, NLRP3, JAK1, and the lysosomal regulator, TFEB. 4-OI and DI are more electrophilic, and are therefore stronger NRF2 activators, and inhibit the production of Type I IFNs, while itaconate inhibits SDH and the dioxygenase, TET2. Additionally, both itaconate and derivates have been shown to be protective across a wide range of mouse models of inflammatory and infectious diseases, through both distinct and overlapping mechanisms. As such, continued research involving the comparison of itaconate and related molecules holds exciting prospects for the study of cysteine modification and pathways for immunomodulation and the potential for new anti-inflammatory therapeutics.

How neutrophil metabolism affects bacterial killing.

Neutrophils are front line cells in immunity that quickly recognize and eliminate pathogens, relying mainly on glycolysis to exert their killing functions. Even though investigations into the influence of metabolic pathways in neutrophil function started in the 1930s, the knowledge of how neutrophils metabolically adapt during a bacterial infection remains poorly understood. In this review, we discuss the current knowledge about the metabolic regulation underlying neutrophils response to bacterial infection. Glycogen metabolism has been shown to be important for multiple neutrophil functions. The potential contribution of metabolic pathways other than glycolysis, such as mitochondrial metabolism, for neutrophil function has recently been explored, including fatty acid oxidation in neutrophil differentiation. Complex III in the mitochondria might also control glycolysis via glycerol-3-phosphate oxidation. Future studies should yield new insights into the role of metabolic change in the anti-bacterial response in neutrophils.

Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses.

Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as Mycobacterium tuberculosis. By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here, we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.