Pyruvate affects inflammatory responses of macrophages during influenza A virus infection

Abstract

Pyruvate is the end product of glycolysis and transported into the mitochondria for use in the tricarboxylic acid (TCA) cycle. It is also a common additive in cell culture media. We discovered that inclusion of sodium pyruvate in culture media during infection of mouse bone marrow derived macrophages with influenza A virus impaired cytokine production (IL-6, IL-1β, and TNF-α). Sodium pyruvate did not inhibit viral RNA replication. Instead, the addition of sodium pyruvate alters cellular metabolism and diminished mitochondrial reactive oxygen species (ROS) production and lowered immune signaling. Overall, sodium pyruvate affects the immune response produced by macrophages but does not inhibit virus replication.

Keywords: Antioxidant; Inflammasome; Inflammation; Influenza A virus; Pyruvate.

Copyright © 2020 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Pyruvate inhibits cytokine responses but not virus replication. BMDM were mock infected (untreated = UT) or infected with 10MOI influenza A/PR8/34/H1N1 virus (IAV) in the presence or absence of sodium pyruvate (NaPyr). After 24 h, cell culture supernatants were collected and examined for cytokine expression by ELISA (A–C) or examined for virus titer by plaque assay (D). Total RNA was isolated from BMDM at the indicted time points after IAV infection in the presence or absence of NaPyr. RNA was transcribed into cDNA and qRT-PCR performed for the indicated viral genes (E–F). BMDM were infected for 24 h in the presence or absence of NaPyr and stained with Sytox-Red then examined by flow cytometry for percentage of cell death (G). Data are representative of 2–4 independent experiments with n = 2–3 wells per experiment. Statistical significance was determined using the students T-test for single comparisons, one-way ANOVA with Tukey post-hoc for multiple comparisons. *** p

Fig. 2

Pyruvate inhibits immune signaling during IAV infection. BMDM were mock infected (untreated = UT) or infected with 10MOI IAV with our without NaPyr. Total RNA was isolated from BMDM at the indicted time points after IAV infection in the presence or absence of NaPyr. RNA was transcribed into cDNA and qRT-PCR performed for the indicated cytokine genes (A–D). Cell lysates were collected at the indicated time points after IAV infection of BMDM in the presence or absence of NaPyr and examined by western blotting for NF-κB activation (phosphorylated-IκBα) (E–F) or caspase-1 activation (Casp-1p20) after 24 h (G–H). Data are representative of 2–4 independent experiments with n = 2–3 wells per experiment. Statistical significance was determined using the students T-test for single comparisons, one-way ANOVA with Tukey post-hoc for multiple comparisons. * p

Fig. 3

Immune responses to LPS + ATP are not affected by NaPyr. BMDM were mock treated (untreated = UT) or treated with 1μM LPS for 3.5 h and then treated with 5 mM ATP for 0.5 h in the presence or absence of NaPyr. Culture supernatants and cell lysates were collected after the total 4 h treatment and examined by ELISA for cytokine production (A–C) or by western blot for caspase-1 activation (Casp-1p20) (D–E). BMDM were mock treated (untreated = UT) or treated with 25μM poly I:C (PIC) in the presence or absence of NaPyr (F). Data are representative of 2–4 independent experiments with n = 2–3 wells per experiment. Statistical significance was determined using the students T-test test for single comparisons, one-way ANOVA with Tukey post-hoc for multiple comparisons. No results were significantly different.

Fig. 4

Altered immune signaling is associated with ROS and ATP levels. BMDM were mock infected (untreated = UT) or infected with 10MOI IAV for 24 h or treated with 1μM LPS for 3.5 h and then treated with 5 mM ATP for 0.5 h, in the presence or absence of NaPyr. Cells were then stained with MitoSox and mitochondrial ROS levels determined by flow cytometry (MFI = Median Fluorescence Intensity) (A–C). Cell supernatants were examined for lactate (D) and lysates were also examined for ATP levels (E). Data are representative of 3–4 independent experiments with n = 2–3 wells per experiment. Statistical significance was determined using the students T-test for single comparisons, one-way ANOVA with Tukey post-hoc for multiple comparisons. * p