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.

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.

Gasdermin D inhibition prevents multiple organ dysfunction during sepsis by blocking NET formation.

Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.

The DNA sensor AIM2 mediates psoriasiform inflammation by inducing type 3 immunity.

Psoriasis is a chronic and recurrent inflammatory skin disease characterized by abnormal proliferation and differentiation of keratinocytes and activation of immune cells. However, the molecular driver that triggers this immune response in psoriatic skin remains unclear. The inflammation-related gene absent in melanoma 2 (AIM2) was identified as a susceptibility gene/locus associated with psoriasis. In this study, we investigated the role of AIM2 in the pathophysiology of psoriasis. We found elevated levels of mitochondrial DNA in patients with psoriasis, along with high expression of AIM2 in both the human psoriatic epidermis and a mouse model of psoriasis induced by topical imiquimod (IMQ) application. Genetic ablation of AIM2 reduced the development of IMQ-induced psoriasis by decreasing the production of type 3 cytokines (such as IL-17A and IL-23) and infiltration of immune cells into the inflammatory site. Furthermore, we demonstrate that IL-17A induced AIM2 expression in keratinocytes. Finally, the genetic absence of inflammasome components downstream AIM2, ASC, and caspase-1 alleviated IMQ-induced skin inflammation. Collectively, our data show that AIM2 is involved in developing psoriasis through its canonical activation.

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.

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.

Pyruvate kinase M2 mediates IL-17 signaling in keratinocytes driving psoriatic skin inflammation.

Psoriasis is an inflammatory skin disease characterized by keratinocyte proliferation and inflammatory cell infiltration induced by IL-17. However, the molecular mechanism through which IL-17 signaling in keratinocytes triggers skin inflammation remains not fully understood. Pyruvate kinase M2 (PKM2), a glycolytic enzyme, has been shown to have non-metabolic functions. Here, we report that PKM2 mediates IL-17A signaling in keratinocytes triggering skin psoriatic inflammation. We find high expression of PKM2 in the epidermis of psoriatic patients and mice undergoing psoriasis models. Specific depletion of PKM2 in keratinocytes attenuates the development of experimental psoriasis by reducing the production of pro-inflammatory mediators. Mechanistically, PKM2 forms a complex with Act1 and TRAF6 regulating NF-κB transcriptional signaling downstream of the IL-17 receptor. As IL-17 also induces PKM2 expression in keratinocytes, our findings reveal a sustained signaling circuit critical for the psoriasis-driving effects of IL-17A, suggesting that PKM2 is a potential therapeutic target for psoriasis.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells.

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.