The IFN-inducible GTPase IRGB10 regulates viral replication and inflammasome activation during influenza A virus infection in mice

Abstract

The upregulation of interferon (IFN)-inducible GTPases in response to pathogenic insults is vital to host defense against many bacterial, fungal, and viral pathogens. Several IFN-inducible GTPases play key roles in mediating inflammasome activation and providing host protection after bacterial or fungal infections, though their role in inflammasome activation after viral infection is less clear. Among the IFN-inducible GTPases, the expression of immunity-related GTPases (IRGs) varies widely across species for unknown reasons. Here, we report that IRGB10, but not IRGM1, IRGM2, or IRGM3, is required for NLRP3 inflammasome activation in response to influenza A virus (IAV) infection in mice. While IRGB10 functions to release inflammasome ligands in the context of bacterial and fungal infections, we found that IRGB10 facilitates endosomal maturation and nuclear translocation of IAV, thereby regulating viral replication. Corresponding with our in vitro results, we found that Irgb10^-/- mice were more resistant to IAV-induced mortality than WT mice. The results of our study demonstrate a detrimental role of IRGB10 in host immunity in response to IAV and a novel function of IRGB10, but not IRGMs, in promoting viral translocation into the nucleus.

Keywords: GTPases; IRGB10; Inflammasome; Influenza A virus; NLRP3; Pyroptosis.

Fig. 1.

IRGB10 facilitates NLRP3 inflammasome activation during IAV infection, while IRGMs are dispensable (A) Microarray gene expression analysis of IFN-stimulated genes in Ifnar1^–/– and Irf1^–/– BMDMs after 9 h of IAV infection [31,54]. (B) mRNA expression of IFN-inducible GTPases 9 h after IAV infection in wild-type, Ifnar1^–/–, and Irf1^–/– BMDMs. (C) Immunoblot analysis of CASP1 cleavage after overnight (16 h) IAV infection in BMDMs lacking IRGB10 or IRGM1/2/3, or NLRP3 as a negative control. (D) ELISA analysis of IL-1β and IL-18 levels in the supernatant of IAV-infected BMDMs 16 hpi. Data are shown as mean + SEM, and significance was determined by a two-tailed unpaired t test (B), ns, not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data shown are from one experiment and are representative of at least three independent experiments.

Fig. 2.

IRGB10 mediates NLRP3 priming and innate signaling (A) Immunoblot analysis of phosphorylated and total IκBα, phosphorylated and total ERK, IRF1, phosphorylated and total STAT1, NLRP3, pro–IL-1β, and β-actin after IAV infection in wild-type and Irgb10^–/– BMDMs. (B) mRNA expression of Nlrp3, pro-Il1b, Il6, Tnfa, Ifng, and Ifnb after IAV infection in wild-type and Irgb10^–/– BMDMs. Data are shown as mean ± SEM, n = 4 and significance was determined by a two-tailed unpaired t test (B) *P < 0.05, **P < 0.01, ***P < 0.001. Data shown are from one experiment and are representative of at least three independent experiments.

Fig. 3.

IRGB10 facilitates viral replication and trafficking of IAV to the nucleus (A) Immunoblot analysis of protein expression of IAV NS1 or IAV NP proteins in wild-type and Irgb10^–/– BMDMs. (B) mRNA expression of viral M1 and Np proteins in wild-type and Irgb10^–/– BMDMs after IAV infection. (C) Plaque-forming units from the supernatant of wild-type and Irgb10^–/– BMDMs 8 h after infection. (D) Uptake of rhodamine-labeled IAV by wild-type and Irgb10^–/–BMDMs. Scale bars indicate 100 μm. (E) Quantification of rhodamine-IAV⁺ BMDMs in wild-type and Irgb10^–/– BMDM 2 h after infection. (F) Localization of IAV NP in wild-type and Irgb10^–/– BMDMs at the indicated timepoints post-infection with IAV. Scale bars indicate 10 μm. (G) Quantification of NP⁺ nuclei in wild-type and Irgb10^–/– BMDM 2 h after infection. Data are shown as mean ± SEM, and significance was determined by a two-tailed unpaired t test (B, C, E, G), *P < 0.05, **P < 0.01, ***P < 0.001. Data shown are from one experiment and are representative of at least three independent experiments.

Fig. 4.

Loss of IRGB10 impairs maturation of IAV-containing endosomes (A) Acidification of pH-rhodamine (pHrodo)-labeled IAV in wild-type or Irgb10^–/– BMDMs. Scale bars indicate 30 μm. (B) Quantification of integrated intensity of pHrodo-labeled IAV 2 h post-infection. Data are shown as mean + SEM, and significance was determined by a two-tailed unpaired t test, ***P < 0.001. Data shown are from one experiment and are representative of at least three independent experiments.

Fig. 5.

IRGB10 enhances susceptibility to IAV infection in vivo Survival of wild-type and Irgb10^–/– mice after IAV infection. Data are pooled from two independent experiments.