Influenza virus activates inflammasomes via its intracellular M2 ion channel

Abstract

Influenza virus, a negative-stranded RNA virus that causes severe illness in humans and animals, stimulates the inflammasome through the Nod-like receptor NLRP3. However, the mechanism by which influenza virus activates the NLRP3 inflammasome is unknown. Here we show that the influenza virus M2 protein, a proton-selective ion channel important in viral pathogenesis, stimulates the NLRP3 inflammasome pathway. M2 channel activity was required for the activation of inflammasomes by influenza and was sufficient to activate inflammasomes in primed macrophages and dendritic cells. M2-induced activation of inflammasomes required its localization to the Golgi apparatus and was dependent on the pH gradient. Our results show a mechanism by which influenza virus infection activates inflammasomes and identify the sensing of disturbances in intracellular ionic concentrations as a previously unknown pathogen-recognition pathway.

Figure 1

Influenza viruses are specifically capable of inducing inflammasome activation. (a) Wild-type BMM were infected with A/PR8, A/Yamagata, A/Beijing, A/Aichi, A/Sydney, A/Guizhou-X, B/Ibaraki, SeV, or HSV-2 at MOI of 2.5. Supernatants were collected 24 h after infection and analyzed for IL-1β, IL-18, and IL-6 by ELISA. (b) Wild-type BMM were infected with A/PR8 (closed circle) or HSV-2 (open circle) at the indicated MOIs. Supernatants were collected 24 h after infection and analyzed for IL-1β and TNF-α by ELISA. Data represent the mean ± S.D. Similar results were obtained from three separate experiments.

Figure 2

Influenza virus activates signal 1 through TLR7. (a, b) BMM prepared from WT, TLR7^−/−, or MAVS^−/− mice were infected with A/Udorn virus. Expression levels of pro-IL-1β were assessed by RT quantitative PCR 24 h after infection (a). Supernatants were collected 24 h after stimulation and analyzed for IL-1β by ELISA (b). (c–e) BMM prepared from WT mice were infected with A/PR8 virus or transfected with CIP-treated, or non-treated A/PR8 genomic RNA or poly (I:C) with DOTAP. Supernatants were collected 24 h after stimulation and analyzed for IL-1β by ELISA (c). Supernatants were analyzed for the presence of mature IL-1β and cell extracts, for the presence of mature IL-β and pro-IL-1β by Western blotting (d). (e) Six hours after infection, total RNA was extracted from virus-infected and RNA-transfected BMM. IFN-β mRNA levels were assessed by RT quantitative PCR. GAPDH was used as an internal control. Data represent the mean ± S.D. Similar results were obtained from three separate experiments.

Figure 3

M2 channel activity of influenza virus is required for inflammasome activation. Cells were infected with WT (A/Udorn) or M2del29-31 (A/Udorn) (a–d) virus. (a) Supernatants from BM DCs infected at indicated MOIs were collected 24 h after infection and analyzed for IL-1β, IL-18, IL-12p40, and TNF-α by ELISA. (b) Supernatants and cell extracts from infected BMM were collected at the indicated time points and analyzed for the presence of pro- and mature-caspase-1 by Western blotting. (c) Pro-IL-1β mRNA levels from infected BMM were assessed by RT quantitative PCR. (d) Supernatants and cell extracts from infected BMM were collected 24 h after infection and the presence of pro- and mature IL-1β were analyzed by Western blotting. Data represent the mean ± S.D, and are representative of at least three independent experiments.

Figure 4

Ectopic expression of M2 channel rescues IL-1β production from M2del29-31 virus infected cells. BMM and BM DC transduced with A/PR8 M2- or GFP-expressing lentivirus were infected with WT or M2del29-31 A/Udorn virus. Supernatants were collected 24 h after infection and analyzed for IL-1β by ELISA. Data represent the mean ± S.D, and are representative of at least three independent experiments.

Figure 5

M2 is sufficient to trigger signal 2 for inflammasome activation. (a) Schematic diagram of influenza virus M2 protein. The amino acid sequence of the transmembrane domain (residues 25 to 43) is shown in the expanded section of the diagram. (b) BMM or BM DC were infected with M2- or GFP-expressing lentivirus in the presence or absence of LPS (50 ng/ml). Supernatants were collected at 24 h post infection and analyzed for IL-1β, IL-18, and TNF. (c) BMM were infected with M2- or GFP-expressing lentiviruses in the presence or absence of LPS (50 ng/ml), and analyzed for the presence of pro- or mature IL-1β by immunoblotting. Data represents the mean ± S.D., and are representative of at least three independent experiments.

Figure 6

Influenza virus M2 protein stimulates IL-1β production from HSV-2 or SeV infected cells. BM DCs were infected with influenza, HSV-2 or SeV in combination with M2- or GFP-lentiviruses. A/PR8 virus was used as a positive control. Supernatants were collected at 24 h post infection and analyzed for IL-1β. Data represents the mean ± S.D., and are representative of at least three independent experiments. *, p < 0.05; **, p < 0.01 compared to non-transduced cells or as indicated, as determined by ANOVA.

Figure 7

M2 triggers inflammasomes through perturbation of ionic homeostasis of the Golgi. (a) BMM were stimulated with A/PR8, HSV-2, SeV, or LPS and cultured in the presence or absence of Monensin (10 µM). (b) BMM were stimulated with A/PR8 virus or LPS + ATP in the presence or absence of Brefeldin A (10µg/ml). Supernatants were collected at 24 h (A/PR8) or 6 h (LPS + ATP) post stimulation and analyzed for IL-1β by ELISA. (c) BSC-1 cells that stably produce the resident Golgi enzyme N-acetylgalactosaminyltransferase II fused to yellow fluorescent protein (GalNAc-T2-YFP) (green) were infected with A/PR8 virus in the presence or absence of Monensin or Brefeldin A. Cells were stained with M2-specific antibody (red) and analyzed by confocal microscopy.

Figure 8

M2 channel-induced inflammasome activation requires acidified Golgi compartment. (a) BMM and BM DC were infected with wild-type or M2del29-31 A/Udorn virus. Six hours later, culture media were replaced with media with the indicated pH levels. Green arrows indicate cytosolic pH level of 7.2. (b) RAW264.7 cells transduced with E5 WT, E5 Q17G, or GFP-expressing retroviruses were infected with A/PR8 at the indicated MOIs. Supernatants were analyzed for IL-1β by ELISA 24 h after infection. Data represents the mean ± S.D. Similar results were obtained from at least three separate experiments.