Mint3/Apba3 depletion ameliorates severe murine influenza pneumonia and macrophage cytokine production in response to the influenza virus

Abstract

Influenza virus (IFV) infection is a common cause of severe pneumonia. Studies have suggested that excessive activation of the host immune system including macrophages is responsible for the severe pathologies mediated by IFV infection. Here, we focused on the X11 protein family member Mint3/Apba3, known to promote ATP production via glycolysis by activating hypoxia inducible factor-1 (HIF-1) in macrophages, and examined its roles in lung pathogenesis and anti-viral defence upon IFV infection. Mint3-deficient mice exhibited improved influenza pneumonia with reduced inflammatory cytokines/chemokine levels and neutrophil infiltration in the IFV-infected lungs without alteration in viral burden, type-I interferon production, or acquired immunity. In macrophages, Mint3 depletion attenuated NF-κB signalling and the resultant cytokine/chemokine production in response to IFV infection by increasing IκBα and activating the cellular energy sensor AMPK, respectively. Thus, Mint3 might represent one of the likely therapeutic targets for the treatment of severe influenza pneumonia without affecting host anti-viral defence through suppressing macrophage cytokine/chemokine production.

Figure 1. Loss of Mint3 attenuates severe influenza pneumonia.

(a) Survival rate of mice after IFV infection. WT and Mint3^−/− mice (n = 22 per group) were intranasally infected with 10⁴ PFU of IFV, followed by the assessment of survival rates. *P < 0.05 by the log-rank test. (b) Hematoxylin and eosin staining of the lungs on days 0, 4, and 8 after IFV infection. Bar = 50 μm. (c,d) Analysis of the BALF collected from WT and Mint3^−/− mice (n = 6 per group) on day 0, 4, and 8 after IFV infection for infiltrated cells (c) and inflammatory cytokine/chemokine levels (d). Infiltrated cells in BALF were analysed for T cells (CD45⁺ CD3ε⁺), B cells (CD45⁺ CD19⁺), natural killer (NK) cells (CD45⁺ NK1.1⁺), neutrophils (CD45⁺ Ly-6G⁺ F4/80⁻), and macrophages (CD45⁺ Ly-6G⁻ F4/80⁺) by flow cytometry and the numbers of total and specific cell populations per lung were calculated. Cytokine/chemokine levels in BALF were measured using a multiplex assay. Data are presented as the means ± SD and are representative of two independent experiments. *P < 0.05, **P < 0.01 by the Student’s t-test.

Figure 2. Mint3 deficiency does not affect the induction of type-I interferons and IFV-specific antibody production.

(a) Viral copy numbers in the infected lungs. The lungs of WT and Mint3^−/− mice (n = 6 per group) at day 0, 4, and 8 following infection with IFV were homogenized. Total RNA was extracted from the infected lungs and viral genome RNA copies were quantified by qPCR. (b) IFN-α and IFN-γ levels in the BALF from WT and Mint3^−/− mice (n = 6 per group) after IFV infection. Data are presented as the means ± SD and are representative of three independent experiments. *P < 0.05, **P < 0.01 by the Student’s t-test. (c) IFV-specific antibody production. WT and Mint3^−/− mice (n = 6 per group) were intranasally infected twice with 10³ PFU of IFV on day 0 and 21. IFV-specific IgG in serum and IgA in the BALF were analysed on day 0 and 7 after the second infection. Data are presented as the means ± SD of triplicates. Data are representative of two independent experiments. *P < 0.05, **P < 0.01 by the Student’s t-test.

Figure 3. Mint3-mediated signalling controls inflammatory cytokine production in response to IFV in MFs but not DCs.

(a–e) AMFs (a), TG-MFs (b), BMMFs (c), cDCs (d), or FLT3L-DCs (e) prepared from WT or Mint3^−/− mice were stimulated in vitro with 10⁶ PFU IFV (M.O.I = 10) for 24 h. IL-6, TNF-α, and CCL2/MCP-1 production in the cell culture supernatants was measured by ELISA. Data are presented as the means ± SD of triplicates and are representative of two independent experiments. *P < 0.05, **P < 0.01 by the Student’s t-test.

Figure 4. Mint3 depletion affects NF-κB activation in BMMFs.

(a) Il6, Tnf, and Ccl2 mRNA expression in WT or Mint3^−/− BMMFs after IFV infection. (b,c) NF-κB expression in nuclear extracts (b) and NF-κB signalling-related factor expression in cytoplasmic extracts (c) of WT or Mint3^−/− BMMFs after IFV infection. Data are presented as the means ± SD of triplicates and are representative of two independent experiments. *P < 0.05, **P < 0.01 by the Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. S3.

Figure 5. Loss of Mint3 affects NF-κB signalling via both AMPK activation and IκB accumulation in BMMFs.

(a) ATP content of BMMFs after IFV infection. (b) AMPK expression in cellular extracts of WT or Mint3^−/− BMMFs after IFV infection. (c) BMMFs were pretreated for 1 h with control vehicle (−), 2-DG (100 μg/mL), oligomycin (5 μg/mL), or AICAR (1 mM) and then infected with 10⁶ PFU (M.O.I = 10) IFV for 24 h. IL-6, TNF-α, and CCL2/MCP-1 production in the cell culture supernatants was measured by ELISA. (d,e) IκBα and AMPK expression in cytoplasmic extracts (d) and NF-κB expression in nuclear extracts (e) of WT or 2-DG-treated WT BMMFs after stimulation with IFV. (f,g) IκBα and AMPK expression in cytoplasmic extracts (f) and NF-κB expression in nuclear extracts (g) of WT or BAY 11-7082-treated WT BMMFs after stimulation with IFV. (h) BMMFs were pretreated for 1 h with control vehicle (−) or BAY 11-7082 (15 μM) and then infected with 10⁶ PFU (M.O.I = 10) IFV for 24 h. IL-6, TNF-α, and CCL2/MCP-1 production in the cell culture supernatants was measured by ELISA. Data are presented as the means ± SD of triplicates and are representative of two independent experiments. **P < 0.05, **P < 0.01 by the Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Fig. S3.

Figure 6. Schematic illustration showing the means by which Mint3 depletion attenuates inflammatory cytokine/chemokine production in IFV-infected macrophages.

Mint3 depletion both activates AMPK and promotes IκBα accumulation in macrophages. These mechanisms contribute to inhibiting the nuclear translocation of NF-κB and the resulting inflammatory cytokine/chemokine production in IFV-infected macrophages.