4-octyl itaconate and itaconate in the regulation of type I interferon in macrophages

Abstract

Itaconate is an endogenous immunomodulatory metabolite produced by the enzyme aconitate decarboxylase 1 (ACOD1), a product of the gene immune-responsive gene 1 (Irg1). Itaconate is the most highly upregulated metabolite after stimulation with the gram-negative bacterial product lipopolysaccharide (LPS) in inflammatory macrophages.

Itaconate is anti-inflammatory, antiviral, and antimicrobial and has been investigated in many inflammatory disease models. Its immunomodulatory potential has gathered significant interest, which aims to use itaconate derivatives and ACOD1 modulators as therapies for inflammatory diseases.

Itaconate partially mediates its immunomodulatory properties by covalently modifying cysteine residues on target proteins, affecting protein function. Itaconate, particularly its cell-permeable derivative 4-octyl itaconate (4-OI), has been shown to alkylate NLRP3, inhibiting NLRP3-NEK7 binding and subsequent inflammasome activation. 4-OI also activates NRF2, which translocates to the nucleus and acts as a transcription factor to drive many antioxidant and cytoprotective genes. Furthermore, itaconate mediates its effects via enzyme inhibition, particularly mitochondrial complex II, also known as succinate dehydrogenase (SDH) and ten eleven translocation 2 (TET2) inhibition. Itaconate's inhibition of SDH creates a breakpoint in the tricarboxylic acid (TCA) cycle, a central feature of macrophage metabolic reprogramming, which inhibits IL1β production.

Importantly, itaconate has been shown to boost LPS-induced type I interferons, while Irg1-/- BMDMs produce less type I interferons. However, the itaconate derivative and tool compound, 4-OI, blocks LPS-induced type I interferons and interferon-stimulated genes (ISGs). It is curious, therefore, how itaconate and its proposed tool compound, 4-OI, have opposing effects in this regard. In this thesis, I have investigated how IFNβ is regulated by itaconate and its derivative, 4-OI.

I have found that 4-OI blocked IFN-β and ISGs upon macrophage challenge with LPS and ligands for TLR3,7/8,9, and Stimulator of Interferon genes (STING). Mechanistically, the suppression of LPS-induced IFN-β by 4-OI was shown to be partially NRF2-dependent. Opposite to 4-OI, I have confirmed that itaconate boosts LPS-induced IFNβ. Furthermore, I have demonstrated that SDH inhibition is responsible for this response by utilising SDH inhibitors and SDH-deficient cells, including human SDH-deficient tumours. I have shown that SDH inhibition by itaconate mediates mitochondrial double-stranded RNA (mtdsRNA) release. Mechanistically, mtRNA release depended on the mitochondrial transporter Voltage-dependent anion channel (VDAC). Following this, the mtdsRNA sensors melanoma differentiation-associated protein 5 (MDA5) and retinoic-acid-inducible gene I (RIG-I) are required for IFNβ production in response to SDH inhibition by itaconate. This was linked to a disease model involving SDH-inhibition, SDH-deficient paraganglioma, where I demonstrated that IFNB is upregulated. Inhibition of SDH by itaconate links TCA cycle modulation to type I interferon production via mtRNA.

These results show a clear difference between itaconate and its derivative, 4-OI, on innate immune responses of murine macrophages.

Together, these results further our understanding of the immunoregulatory role of itaconate. I have shown that 4-OI blocks type I interferon via NRF2 and that itaconate boosts LPS-induced type I interferons through SDH inhibition and mtdsRNA release. This thesis further emphasises the role of the TCA cycle in inflammation, demonstrates the complexity of itaconate biology, and highlights the importance of understanding the immunomodulatory mechanisms of itaconate for generating new therapeutics based on itaconate for immunological diseases.