Resumen de Role of the interferon-stimulated gene 15 (ISG15) as a regulator of macrophage metabolism after Vaccinia virus infection
The production of interferons (IFNs) is one of the most relevant and best known antiviral strategies of the innate immune response. IFNs induce the expression of a wide variety of IFN-stimulated genes (ISGs), being Isg15 one of the most highly expressed ISGs. Isg15 encodes the protein ISG15, a ubiquitin-like protein that modulates the cell proteome through non-covalent and covalent interactions with target proteins. ISG15 is also secreted out of the cell, acting as a cytokine that regulates immune cell functions. ISG15 was first identified as an antiviral molecule, but research along the last two decades has demonstrated that ISG15 participates in the regulation of diverse cellular pathways, from genome replication to energy metabolism. In this work, we explored the role of ISG15 in the regulation of macrophage metabolism, as well as the relevance of ISG15 in the interaction between macrophages and Vaccinia virus (VACV) from a metabolic point of view. For that purpose, we used a proteomics and lipidomics-based approach, which revealed alterations in mitochondrial and lipid metabolism in Isg15-/-bone marrow-derived macrophages (BMDM). Our results demonstrated that Isg15-/-BMDM display impaired OXPHOS and ROS production, increased mitochondrial fusion, and defective mitophagy, concomitant with accumulation of mitochondrial proteins. Regarding lipid metabolism, Isg15-/-BMDM showed increased levels of proteins involved in lipid hydrolysis and oxidation compared with Isg15+/+ BMDM, consistent with a strong decrease in neutral lipid levels, reflected by reduced lipid droplet number and size. Such changes, however, were not linked to increased fatty acid oxidation. VACV provoked a marked inhibition of both Isg15 expression and ISG15 conjugation to target proteins, causing similar mitochondrial alterations to those observed in Isg15-/-BMDM. As well, VACV-infected BMDM showed increased levels of proteins involved in lipid uptake and storage, consistent with higher neutral lipid content compared with uninfected cells. The alterations on lipid metabolism caused by VACV were stronger in Isg15-/-BMDM, suggesting that ISG15 is required to restrict the metabolic reprogramming induced by VACV infection. Furthermore, both the absence of ISG15 and VACV infection of BMDM led to the development of a mixed activation phenotype, showing features of both M1 and M2 polarization. Last, we investigated the role of ISG15 in the regulation of VACV morphogenesis and spread in mouse embryonic fibroblasts (MEF), and detected a reduction in actin tail formation and extracellular virus release in Isg15-/MEF. This was consistent with the accumulation of intracellular virus particles, the abrogation of comet-shaped plaques, and changes in the virion proteome of viruses purified from Isg15-/-or Isg15+/+ MEF. Overall, this work introduces ISG15 as a novel regulator of macrophage metabolism, and highlights its importance in the interplay between VACV and the host
