Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection

Abstract

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. Itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

“…In addition, the consequences of ACOD1 inhibition may become fully manifest only at the organism level and may not be apparent in cellular models. Indeed, we recently found that CXCL10 expression in IAV-infected bone marrow-derived macrophages did not differ between wild-type and ACOD1 –/– mice, whereas histologically assessed pulmonary inflammation was significantly higher in ACOD1 –/– mice 10 . In contrast to inflammation, low-dose citraconate did have a marked effect on TCA cycle intermediates and tended to improve mitochondrial respiration in dTHP1 cells stimulated with LPS/IFN-γ.…”

“…Itaconate derivatives are being studied as antiviral compounds and adjunct treatments to modify host responses in viral infections 9 , 10 . We therefore infected dTHP1 and A549 cells with IAV and treated them with non-toxic concentrations of the three isomers, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Supplementary Fig.…”

“…171-A1112) as described in ref. 10 , from which the following text was used: “Standard curves were generated with eight 2-fold serial dilutions starting from 32 ng/mL. 100 μL of assay buffer was added to each well of a microfiltration plate, followed by addition of 50 μL of beads suspension.…”

“…Explanted lung tissue from patients with a clinical indication for lung transplantation was divided into pieces of approximately 30 mg and cultured and infected essentially as described in ref. 10 . Tissues were kept in RPMI buffer for up to 20 h before the start of the experiment.…”

“…The immunomodulatory and cytoprotective properties of itaconate and chemically modified derivatives such as dimethyl- and 4-octyl-itaconate (4-OI) are being studied intensely to create therapeutic interventions for inflammatory and degenerative diseases 3 , 4 and viral infections 9 , 10 . By contrast, it is not known whether exogenously applied mesaconate or citraconate can be taken up by cells and whether they exert immunomodulatory, anti-oxidative or anti-infective effects similar to itaconate.…”