Itaconate drives mtRNA-mediated type I interferon production through inhibition of succinate dehydrogenase.

Itaconate is one of the most highly upregulated metabolites in inflammatory macrophages and has been shown to have immunomodulatory properties. Here, we show that itaconate promotes type I interferon production through inhibition of succinate dehydrogenase (SDH). Using pharmacological and genetic approaches, we show that SDH inhibition by endogenous or exogenous itaconate leads to double-stranded mitochondrial RNA (mtRNA) release, which is dependent on the mitochondrial pore formed by VDAC1. In addition, the double-stranded RNA sensors MDA5 and RIG-I are required for IFNß production in response to SDH inhibition by itaconate. Collectively, our data indicate that inhibition of SDH by itaconate links TCA cycle modulation to type I interferon production through mtRNA release.

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.

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.

Targeting immunometabolism to treat COVID-19.

The COVID-19 crisis has emphasised the need for antiviral therapies to combat current and future viral zoonoses. Recent studies have shown that immune cells such as macrophages are the main contributors to the inflammatory response seen in the later inflammatory phase of COVID-19. Immune cells in the context of a viral infection such as SARS-CoV-2 undergo metabolic reprogramming to elicit these pro-inflammatory effector functions. The evidence of metabolic reprogramming in COVID-19 offers opportunities for metabolites with immunomodulatory properties to be investigated as potential therapies to combat this hyper-inflammatory response. Recent research indicates that the metabolite itaconate, previously known to be broadly antibacterial, may have both antiviral and immunomodulatory potential. Furthermore, low itaconate levels have shown to correlate with COVID-19 disease severity, potentially implicating its importance in the disease. The antiviral potential of itaconate has encouraged researchers to synthesise itaconate derivatives for antiviral screening, with some encouraging results. This review summarises the antiviral and immunomodulatory potential of immunometabolic modulators including metformin, peroxisome proliferator-activated receptor agonists and TEPP-46 as well as itaconate, and its derivatives and their potential use as broad spectrum anti-viral agents.

Extracellular Ca2+-Sensing Fluorescent Protein Biosensor Based on a Collagen-Binding Domain.

The importance of extracellular gradients of biomolecules is increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, the dynamics of extracellular calcium concentration is rarely studied. Here, we present a low affinity Ca2+ biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable scaffold-based biosensing of Ca2+ in the proximity of cells in live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one- and two-photon excitation modes. Furthermore, the donor fluorescence lifetime of the biosensor displays response to [Ca2+] over a range of ∼2-2.5 ns, making it attractive for multiplexed FLIM assays. To evaluate the performance of this biosensor in physiological measurements, we applied it to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca2+ upon chelation with EGTA. When combined with spectrally resolved FLIM of lipid droplets using Nile red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca2+ depletion, suggesting that the intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate Twitch-2B as a prospective Ca2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment.