Treatment with siRNAs is commonly associated with GPX4 up-regulation and target knockdown-independent sensitization to ferroptosis.

Small interfering RNAs (siRNAs) are widely used in biomedical research and in clinical trials. Here, we demonstrate that siRNA treatment is commonly associated with significant sensitization to ferroptosis, independently of the target protein knockdown. Genetically targeting mitochondrial antiviral-signaling protein (MAVS) reversed the siRNA-mediated sensitizing effect, but no activation of canonical MAVS signaling, which involves phosphorylation of IkBα and interferon regulatory transcription factor 3 (IRF3), was observed. In contrast, MAVS mediated a noncanonical signal resulting in a prominent increase in mitochondrial ROS levels, and increase in the BACH1/pNRF2 transcription factor ratio and GPX4 up-regulation, which was associated with a 50% decrease in intracellular glutathione levels. We conclude that siRNAs commonly sensitize to ferroptosis and may severely compromise the conclusions drawn from silencing approaches in biomedical research. Finally, as ferroptosis contributes to a variety of pathophysiological processes, we cannot exclude side effects in human siRNA-based therapeutical concepts that should be clinically tested.

vPIF-1 is an insulin-like antiferroptotic viral peptide.

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.

Genome Replication Is Associated With Release of Immunogenic DNA Waste.

Innate DNA sensors detect foreign and endogenous DNA to induce responses to infection and cellular stress or damage. Inappropriate activation by self-DNA triggers severe autoinflammatory conditions, including Aicardi-Goutières syndrome (AGS) that can be caused by defects of the cytosolic DNase 3'repair exonuclease 1 (TREX1). TREX1 loss-of-function alleles are also associated with systemic lupus erythematosus (SLE). Chronic activation of innate antiviral immunity in TREX1-deficient cells depends on the DNA sensor cGAS, implying that accumulating TREX1 DNA substrates cause the inflammatory pathology. Retrotransposon-derived cDNAs were shown to activate cGAS in TREX1-deficient neuronal cells. We addressed other endogenous sources of cGAS ligands in cells lacking TREX1. We find that induced loss of TREX1 in primary cells induces a rapid IFN response that requires ongoing proliferation. The inflammatory phenotype of Trex1-/- mice was partially rescued by additional knock out of exonuclease 1, a multifunctional enzyme providing 5' flap endonuclease activity for Okazaki fragment processing and postreplicative ribonucleotide excision repair. Our data imply genome replication as a source of DNA waste with pathogenic potential that is efficiently degraded by TREX1.